Assay devices and methods of manufacture

ABSTRACT

Systems, methods, and apparatuses are provided for self-contained nucleic acid preparation, amplification, and analysis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 national phase application ofInternational Application Serial No. PCT/US2020/057600, filed Oct. 28,2020, which claims the benefit of and priority to U.S. Prov. Pat. App.No. 62/927,481 filed 29 Oct. 2019, which are each incorporated herein byreference in their entirety. This application also references FrenchPatent Application FR1912110 filed 29 Oct. 2019, the entirety of whichis incorporated herein by reference.

BACKGROUND 1. Technical Field

Embodiments of the present disclosure relate generally to test devices,for example, for amplifying nucleic acids and methods of manufacture ofsuch test devices.

2. Background

In the United States, Canada, and Western Europe infectious diseaseaccounts for approximately 7% of human mortality, while in developingregions infectious disease accounts for over 40% of human mortality.Infectious diseases lead to a variety of clinical manifestations. Amongcommon overt manifestations are fever, pneumonia, meningitis, diarrhea,and diarrhea containing blood. While the physical manifestations suggestsome pathogens and eliminate others as the etiological agent, a varietyof potential causative agents remain, and clear diagnosis often requiresa variety of assays be performed. Traditional microbiology techniquesfor diagnosing pathogens can take days or weeks, often delaying a propercourse of treatment.

In recent years, the polymerase chain reaction (PCR) has become a methodof choice for rapid diagnosis of infectious agents. PCR can be a rapid,sensitive, and specific tool to diagnose infectious disease. However, achallenge to using PCR as a primary means of diagnosis is the variety ofpossible causative organisms or viruses and the low levels of organismor virus present in some pathological specimens. It is often impracticalto run large panels of PCR assays, one for each possible causativeorganism or viruses, most of which are expected to be negative. Theproblem is exacerbated when pathogen nucleic acid is at lowconcentration and requires a large volume of sample to gather adequatereaction templates. In some cases there is inadequate sample to assayfor all possible etiological agents. A solution is to run “multiplexPCR” wherein the sample is concurrently assayed for multiple targets ina single reaction. While multiplex PCR has proved to be valuable in somesystems, shortcomings exist concerning robustness of high levelmultiplex reactions and difficulties for clear analysis of multipleproducts. To solve these problems, the assay may be subsequently dividedinto multiple secondary PCRs. Nesting secondary reactions within theprimary product increases robustness. Closed systems such as theFilmArray® (BioFire Diagnostics, LLC, Salt Lake City, Utah) reducehandling, thereby diminishing contamination risk.

The present invention addresses various improvements relating toautomated or semi-automated manufacturing of test devices, cost of testdevices, and more rapid sample-to-answer.

BRIEF SUMMARY

Described herein are self-contained reaction containers (also referredto herein as ‘pouches’ or ‘cards’), methods of manufacturing suchreaction containers, instruments, systems, and methods for rapidamplification of nucleic acids. In an illustrative embodiment, areaction container may be fabricated from a first sheet and a secondsheet of polymeric material with one or more fluidically connectedreaction chambers and reagent reservoirs (e.g., aqueous reagentreservoirs) formed between the first and second sheets. The reactionchambers and reagent reservoirs may be formed by pressing the first andsecond polymeric sheets between forming plates and propelling acompressed fluid between the sheets to form open areas.

Described herein are:

A1. A method for forming a reaction container, comprising:

providing a polymeric sheet that comprises an inner planar face and anouter planar face;

contacting a first inner planar face of the polymeric sheet to a secondinner planar face of the polymeric sheet;

pressing the polymeric sheet between a first forming plate and a secondforming plate, wherein at least one of the first or second formingplates has one or more recesses for forming one or more featuresselected from the group consisting of a reaction chamber, a fluid flowchannel, a reagent chamber, or a sample chamber in selected portions ofthe polymeric sheet;

propelling a compressed fluid between the inner planar faces of thepolymeric sheet while the polymeric sheet is pressed between the formingplates to reform the selected portions of the polymeric sheet into ashape defined by the one or more recesses of the forming plates;

separating the first forming plate and the second forming plate so thepolymeric sheet is no longer pressed between the first and secondforming plates; and

removing the reaction container from between the first forming plate andthe second forming plate.

A2. The method of clause A1, wherein the provided polymeric sheetcomprises a first polymeric sheet and a second polymeric sheet, eachpolymeric sheet having an inner planar face and an outer planar face,and the method further comprising:

arranging the first and second polymeric sheets so that the inner planarfaces are arranged adjacent to one another,

performing the pressing step, and

propelling a compressed fluid between the two polymeric sheets while thetwo polymeric sheets are pressed between the forming plates to reformthe selected portions into a shape defined by the one or more recessesof the forming plates.

A3. The method of any of clauses A1 or A2 further comprising heating thepolymeric sheet to a softening temperature prior to the pressing step,performing the pressing and propelling steps, and cooling the polymericsheet after the propelling step to set the shape defined by the one ormore recesses of the forming plates.

A4. The method of any of clauses A1-A3, wherein the polymeric sheetcomprises a flexible polymeric material having a thickness in a range ofabout 0.02 mm to about 0.1 mm.

A5. The method of any of clauses A1-A4, wherein the flexible polymericmaterial is selected from the group consisting of polyester,polyethylene, polyethylene terephthalate (PET), polycarbonate,polypropylene (PP), polymethylmethacrylate, mixtures, combinationsthereof.

A6. The method of any of clauses A1-A5, wherein the flexible polymericmaterial comprises a water vapor and/or oxygen barrier material.

A7. The method of any of clauses A1-A6, wherein the polymeric materialhas a water vapor transmission rate (WVTR) in a range of about 0.01g/m²/24 hrs to about 3 g/m²/24 hrs, preferably in a range of about 0.05g/m²/24 hrs to about 2 g/m²/24 hrs, or more preferably no more thanabout 1 g/m²/24 hrs, and an oxygen transmission rate in a range of about0.01 cc/m²/24 hrs to about 2 cc/m²/24 hrs, preferably in a range ofabout 0.05 cc/m²/24 hrs to about 2 cc/m²/24 hrs, or more preferably nomore than about 1 cc/m²/24 hrs.

A8. The method of any of clauses A1-A7, wherein the flexible polymericmaterial comprises two or more layers of film material bonded togetherand the water vapor and/or oxygen barrier comprises at least one of ametalized or ceramic-coated film layer.

A9. The method of any of clauses A1-A8, wherein the selected portionscomprise one or more of a sample input chamber, a sample preparationchamber, a sample reactant recovery/wash chamber, a reaction chamber, orone or more fluid reagent reservoirs.

A10. The method of any of clauses A1-A9, further comprising heating thepolymeric sheet to a first temperature prior to the pressing step.

A11. The method of any of clauses A1-A10, wherein separating the firstforming plate and the second forming plate comprises moving at least oneof the first forming plate and the second forming plate.

A12. The method of any of clauses A1-A11, wherein separating the firstforming plate and the second forming plate comprises moving only one ofthe first forming plate and the second forming plate.

B1. A method for forming a reaction container, comprising:

providing a first polymeric sheet and a second polymeric sheet, whereinthe first and second polymeric sheets each comprise an inner planar faceand an outer planar face;

contacting the inner planar face of the first polymeric sheet to theinner planar face of the second polymeric sheet;

laminating the first polymeric sheet to the second polymeric sheet;

making one or more seal lines joining the first and second polymericsheets;

pressing the first and second polymeric sheets between a first formingplate and a second forming plate, wherein at least one of the first orsecond forming plates has one or more recesses positioned for reformingthe first and second polymeric sheets to form one or more openings in aregion defined by the one or more seal lines;

expanding selected areas of the first and second polymeric sheets into ashape defined by the one or more recesses of the forming plates byblowing a compressed gas between the first and second polymeric sheetswhile the first and second polymeric sheets are pressed between theforming plates;

separating the first forming plate and the second forming plate; and

removing the reaction container from between the first forming plate andthe second forming plate.

B2. The method of clause B1, further comprising cooling the first andsecond polymeric sheets subsequent to the expanding step to set theshape defined by the one or more recesses of the forming plates.

B3. The method of any of clauses B1 or B2, wherein the compressed gasblown between the first and second polymeric sheets substantiallysimultaneously expands and cools the first and second polymeric sheets.

B4. The method of any of clauses B1-B3, wherein making the one or moreseal lines comprises defining one or more of a sample input chamber, asample preparation chamber, a sample reactant recovery/wash chamber, atleast one reaction chamber, a one or more fluid reagent reservoirs, orone or more channels fluidically connecting the sample input chamber,the sample preparation chamber, the sample reactant recovery/washchamber, the at least one reaction chamber, and the one or more reagentreservoirs.

B5. The method of any of clauses B1-B4, wherein when the first andsecond polymeric sheets are pressed between a first forming plate and asecond forming plate the one or more recesses of the forming platessubstantially align with the one or more areas defined by the seallines, and the selected areas expanded by blowing a compressed gasbetween the first and second polymeric sheets comprise one or more ofthe sample input chamber, the sample preparation chamber, therecovery/wash chamber, one or more reaction chambers, or one or morereagent reservoirs, and wherein one or more of the selected areasexpanded by blowing the compressed gas between the first and secondpolymeric sheets are connected by one or more sealed, openable laminatedchannels.

B6. The method of any of clauses B1-B5, wherein the reaction containercomprises sample input chamber fluidically connected to a first reactionchamber, a second reaction chamber fluidically connected to the firstreaction chamber, and at least one reagent reservoir fluidicallyconnected to the sample input chamber, the first reaction chamber, orthe second reaction chamber, and wherein the method further comprises:

expanding the sample input chamber and the at least one reagentreservoir into shapes defined by the recesses of the forming plates byblowing the compressed gas between the first and second polymeric sheetswhile the first and second polymeric sheets are pressed between theforming plates.

B7. The method of any of clauses B1-B6, further comprising expanding thesecond reaction chamber into a shape defined by the recesses of theforming plates by blowing the compressed gas between the first andsecond polymeric sheets while the first and second polymeric sheets arepressed between the forming plates.

B8. The method of any of clauses B1-B7, further comprising:

making the one or more seal lines defining a second reaction chamber,

expanding the second reaction chamber into a shape defined by a secondreaction chamber recess of the forming plates,

providing a reaction card having a plurality of wells formed therein andspotted with one or more dried reagents for a second stage reaction,

inserting the reaction card into the second reaction chamber via anopening between the first and second sheets;

bonding a first planar face of the reaction card to the first sheet anda second, opposite planar face of the reaction card to the second sheet,and

sealing the opening used to insert the reaction card by sealing thefirst polymeric sheet to the second polymeric sheet at the opening.

B9. The method of any of clauses B1-B8, further comprising:

injecting a selected aqueous reagent into the at least one reagentreservoir via a reagent reservoir opening between the first and secondpolymeric sheets,

sealing the selected aqueous reagent in the at least one reagentreservoir by sealing the first polymeric sheet to the second polymericsheet at the reagent reservoir opening such that the reaction containeris provided with an aqueous reagent at the time of manufacture.

B10. The method of any of clauses B1-B9, further comprising:

expanding a fluid reservoir and an access channel in the first reactionchamber into shapes defined by the recesses of the forming plates byblowing the compressed gas between the first and second polymeric sheetswhile the first and second polymeric sheets are pressed between theforming plates,

injecting an aqueous reagent into the fluid reservoir in the firstreaction chamber via the access channel, and

sealing the sample preparation reagent in the fluid reservoir in thesample preparation chamber by sealing the first polymeric sheet to thesecond polymeric sheet at the access channel such that the reactioncontainer is provided with the aqueous reagent in the first reactionchamber at the time of manufacture.

B11. The method of any of clauses B1-B10, wherein the first and secondpolymeric sheets comprise a water vapor and/or oxygen barrier material.

B12. The method of any of clauses B1-B11, wherein the first and secondpolymeric sheets have a water vapor transmission rate (WVTR) in a rangeof about 0.01 g/m²/24 hrs to about 3 g/m²/24 hrs, preferably in a rangeof about 0.05 g/m²/24 hrs to about 2 g/m²/24 hrs, or more preferably nomore than about 1 g/m²/24 hrs, and/or an oxygen transmission rate in arange of about 0.01 cc/m²/24 hrs to about 2 cc/m²/24 hrs, preferably ina range of about 0.05 cc/m²/24 hrs to about 2 cc/m²/24 hrs, or morepreferably no more than about 1 cc/m²/24 hrs.

B13. The method of any of clauses B1-B12, wherein the water vapor and/oroxygen barrier material comprises at least one of a metalized orceramic-coated film layer.

B14. The method of any of clauses B1-B13, wherein the first and secondpolymeric sheets comprise a material selected from the group consistingof polyester, polyethylene, polyethylene terephthalate (PET),polycarbonate, polypropylene (PP), polymethylmethacrylate, mixtures,combinations thereof.

B15. The method of any of clauses B1-B14, further comprising:

prior to the laminating step, dispensing droplets of one or more liquidreagents onto the first polymeric sheet or the second polymeric sheetand drying the droplets of liquid reagent dispensed onto the firstpolymeric sheet or the second polymeric sheet,

wherein the droplets of the one or more liquid reagents are dispensedand dried in one or more areas to be formed into the sample inputchamber, the sample preparation chamber, the sample reactantrecovery/wash chamber, the at least one reaction chamber, or the one ormore channels fluidically connecting the sample input chamber, thesample preparation chamber, the sample reactant recovery/wash chamber,the at least one reaction chamber, and the one or more reagentreservoirs.

B16. The method of any of clauses B1-B15, further comprising heating thefirst and second polymeric sheets to a temperature sufficient forreforming first and second polymeric sheets prior to the pressing step.

B17. The method of any of clauses B1-B16, wherein separating the firstforming plate and the second forming plate comprises moving at least oneof the first forming plate and the second forming plate.

B18. The method of any of clauses B1-B17, wherein separating the firstforming plate and the second forming plate comprises moving only one ofthe first forming plate and the second forming plate.

C1. A method for forming a reaction container formed from a first sheetand a second sheet and having a first reaction chamber, a reagentreservoir, and a channel fluidically connecting the reaction chamber andthe reagent reservoir, the method comprising:

laminating the first sheet to the second sheet;

making one or more seal lines joining the first and second sheets todefine the first reaction chamber, the reagent reservoir, and thechannel;

pressing the first and second sheets between a forming die having afirst plate and a second plate, wherein the forming die comprisesrecesses having a shape corresponding to the reagent reservoir;

propelling a fluid between the first and second sheets while the firstand second sheets are clamped in the forming die to reform selectedareas of the first and second sheets into the shapes of the recesses;and

removing the reaction container from the forming die.

C2. The method of clause C1, further comprising injecting an aqueousreagent into the reagent reservoir via a first reagent reservoir openingbetween the first and second sheets, sealing the aqueous reagent in thereagent reservoir by sealing the first reagent reservoir opening suchthat the reaction container is provided with an aqueous reagent at thetime of manufacture.

C3. The method of any of clauses C1 or C2, wherein the reactioncontainer further comprises a second reaction chamber fluidly connectedto the first reaction chamber by a second channel, the method furthercomprising:

making the one or more seal lines to join the first and second sheets todefine the first reaction chamber, the reagent reservoir, the firstchannel, the second reaction chamber, and the second channel, and

performing the clamping and propelling steps to selectively to form thereagent reservoir and the second reaction chamber, wherein the formingdie further comprises a recess having a shape corresponding to thesecond reaction chamber.

C4. The method of any of clauses C1-C3, further comprising:

providing a reaction card having a plurality of wells formed therein andspotted with one or more dried reagents for a second stage reaction,

inserting the reaction card into the second reaction chamber via asecond reaction chamber opening between the first and second sheets;

bonding a first planar face of the reaction card to the first sheet anda second, opposite planar face of the reaction card to the second sheet,and

sealing the second reaction chamber opening.

C5. The method of any of clauses C1-C4, wherein the reaction containerfurther comprises a sample input chamber, a sample preparation chamber,and a sample reactant recovery/wash chamber, upstream of first reactionchamber and a plurality of channels fluidly connecting the sample inputchamber, the sample preparation chamber, and the sample reactantrecovery/wash chamber to the first reaction chamber, the method furthercomprising:

making the one or more seal lines to join the first and second sheets toadditionally define each of the sample input chamber, the samplepreparation chamber, the sample reactant recovery/wash chamber, and theplurality of channels,

performing the clamping and propelling steps to additionally form thesample input chamber, the sample preparation chamber, and the samplereactant recovery/wash chamber, wherein the forming die furthercomprises recesses having shapes corresponding to the sample inputchamber and the sample reactant recovery/wash chamber.

C6. The method of any of clauses C1-05, wherein the forming die furthercomprises recesses positioned and configured for forming a plurality ofreagent reservoirs fluidly connected to the sample preparation chamber,the sample reactant recovery/wash chamber, and the first reactionchamber, and the method further comprising:

making the one or more seal lines to join the first and second sheets toadditionally define each of the plurality of reagent reservoirs and aplurality of channels fluidically connecting them to one or more of thesample input chamber, the sample preparation chamber, the samplereactant recovery/wash chamber, the first reaction chamber, or thesecond reaction chamber;

performing the clamping and propelling steps to additionally form eachof the plurality of reagent reservoirs,

injecting a selected aqueous reagent into each of the plurality ofreagent reservoirs via a plurality of reagent reservoir opening betweenthe first and second sheets,

sealing the aqueous reagents in each of the plurality of reagentreservoirs by sealing the openings such that the reaction container isprovided with a plurality of aqueous reagents at the time ofmanufacture.

C7. The method of any of clauses C1-C6, wherein the forming die furthercomprises a recess having a shape corresponding to a fluid reservoirpositioned in the sample preparation chamber, and the method furthercomprising:

performing the clamping and propelling steps to form the fluid reservoirin the sample preparation chamber,

injecting a sample preparation reagent into the fluid reservoir via asample preparation chamber opening between the first and second sheets,and

sealing the sample preparation reagent in the fluid reservoir in thesample preparation chamber by sealing the sample preparation chamberopening such that the reaction container is provided with samplepreparation reagent at the time of manufacture.

C8. The method of any of clauses C1-C7, wherein the first sheet to thesecond sheet comprise a flexible polymeric material.

C9. The method of any of clauses C1-C8, wherein the flexible polymericmaterial comprises a water vapor and/or oxygen barrier material.

C10. The method of any of clauses C1-C9, wherein the polymeric materialcomprising the water vapor and/or oxygen barrier material has a watervapor transmission rate (WVTR) in a range of about 0.01 g/m²/24 hrs toabout 3 g/m²/24 hrs, preferably in a range of about 0.05 g/m²/24 hrs toabout 2 g/m²/24 hrs, or more preferably no more than about 1 g/m²/24hrs, and an oxygen transmission rate in a range of about 0.01 cc/m²/24hrs to about 2 cc/m²/24 hrs, preferably in a range of about 0.05cc/m²/24 hrs to about 2 cc/m²/24 hrs, or more preferably no more thanabout 1 cc/m²/24 hrs.

C11. The method of any of clauses C1-C10, wherein the polymeric materialis selected from the group consisting of polyester, polyethylene,polyethylene terephthalate (PET), polycarbonate, polypropylene (PP),polymethylmethacrylate, mixtures, combinations thereof.

C12. The method of any of clauses C1-C11, wherein the water vapor and/oroxygen barrier material comprises at least one of a metalized orceramic-coated film layer.

C13. The method of any of clauses C1-C12, wherein making the one or moreseal lines to join the first and second sheets to define the reactionchamber, the reagent blister, and the channel comprises one or more ofheat sealing, sonic welding, or laser welding.

C14. The method of any of clauses C1-C13, wherein the first and secondsheets are heated prior to the clamping step.

C15. The method of any of clauses C1-C14, wherein the heating comprisesselectively heating only regions of the first and second sheets definingthe reaction chamber and the reagent blister.

C16. The method of any of clauses C1-C15, wherein selectively heatingcomprises clamping the first and second sheets in between heated plateshaving raised areas corresponding to the reaction chamber and thereagent blister.

C17. The method of any of clauses C1-C16, wherein the fluid propelledbetween the first and second sheets is compressed air.

C18. The method of any of clauses C1-C17, further comprising forming oneor more holes in the first sheet or the second sheet and one or morechannels in fluid communication with the one or more holes, wherein theone or more holes are in fluid communication with corresponding conduitsin the forming die for propelling the fluid between the first and secondsheets to form the reaction chamber and the reagent blister.

C19. The method of any of clauses C1-C18, wherein the one or more holesin the first sheet or the second sheet are clamped in the forming die influid communication with the conduits for propelling the fluid betweenthe first and second sheets.

C20. The method of any of clauses C1-C19, wherein removing the reactioncontainer from the forming die comprises separating the first formingplate and the second forming plate so the first and second sheets are nolonger clamped in the forming die.

C21. The method of any of clauses C1-C20, wherein separating the firstforming plate and the second forming plate comprises moving at least oneof the first forming plate and the second forming plate.

C22. The method of any of clauses C1-C21, wherein separating the firstforming plate and the second forming plate comprises moving only one ofthe first forming plate and the second forming plate.

C23. The method of any of clauses C1-C22, wherein the one or more holesare in the first sheet only and the conduits are not in fluidcommunication with any holes in the second sheet.

D1. A method for forming a reaction container formed from a first sheetand a second sheet and having a reaction chamber, a reagent reservoir, achannel fluidically connecting the reaction chamber and the reagentreservoir, and one or more dried reagents disposed in the reactioncontainer between the first sheet and the second sheet, the methodcomprising:

dispensing one or more liquid reagents onto the first sheet or thesecond sheet;

drying the liquid reagents dispensed onto the first sheet or the secondsheet;

laminating the first sheet to the second sheet, wherein the laminatingincludes heating the first and second sheets and compressing them, andwherein the laminated first and second sheets are reversibly sealed toone another;

forming one or more seal lines substantially irreversibly bonding thefirst and second sheets together at the seal lines to define thereaction chamber, the reagent reservoir, and the channel;

clamping the first and second sheets in a forming die having a firstplate and a second plate, wherein the forming die comprises a recesshaving a shape corresponding to the reagent reservoir;

propelling a fluid between the first and second sheets while the firstand second sheets are clamped in the forming die to reform selectedareas of the first and second sheets into the shapes of the recesses;and

removing the reaction container from the forming die.

D2. The method of clause D1, wherein the liquid reagents are dispensedonto the first or second sheet as droplets.

D3. The method of any of clauses D1 or D2, wherein the liquid reagentsare water-based.

D4. The method of any of clauses D1-D3, wherein the liquid reagents areair dried on the first or second sheet prior to the laminating.

D5. The method of any of clauses D1-D4, wherein the one or more liquidreagents are dispensed onto the first or second sheet and dried in aregion to be formed into the reaction chamber.

D6. The method of any of clauses D1-D5, further comprising injecting aan aqueous reagent into the reagent blister via an opening between thefirst and second sheets, sealing the fluid reagent in the reactioncontainer by sealing the opening such that the reaction container isprovided with the fluid reagent at the time of manufacture.

D7. The method of any of clauses D1-D6, wherein the aqueous reagent isconfigured for rehydrating the one or more dried reagents disposed inthe reaction container in preparation for performing an assay using thereaction container.

D8. The method of any of clauses D1-D7, wherein the first sheet to thesecond sheet comprise a flexible polymeric material selected from thegroup consisting of polyester, polyethylene, polyethylene terephthalate(PET), polycarbonate, polypropylene (PP), polymethylmethacrylate,mixtures, combinations thereof.

D9. The method of any of clauses D1-D8, wherein the flexible polymericmaterial comprises a water vapor and/or oxygen barrier material.

D10. The method of any of clauses D1-D9, wherein the polymeric materialcomprising the water vapor and/or oxygen barrier material has a watervapor transmission rate (WVTR) in a range of about 0.01 g/m²/24 hrs toabout 3 g/m²/24 hrs, preferably in a range of about 0.05 g/m²/24 hrs toabout 2 g/m²/24 hrs, or more preferably no more than about 1 g/m²/24hrs, and an oxygen transmission rate in a range of about 0.01 cc/m²/24hrs to about 2 cc/m²/24 hrs, preferably in a range of about 0.05cc/m²/24 hrs to about 2 cc/m²/24 hrs, or more preferably no more thanabout 1 cc/m²/24 hrs.

D11. The method of any of clauses D1-D10, wherein the liquid reagentsdispensed onto the first or second sheet comprise an enzyme selected foruse in a molecular biological or immunological assay (e.g., a reversetranscriptase, a DNA polymerase, and combinations thereof).

D12. The method of any of clauses D1-D11, wherein the enzyme regains itsactivity following the drying, laminating, and rehydration.

D13. The method of any of clauses D1-D12, wherein removing the reactioncontainer from the forming die comprises separating the first formingplate and the second forming plate so the first and second sheets are nolonger clamped in the forming die.

D14. The method of any of clauses D1-D13, wherein separating the firstforming plate and the second forming plate comprises moving at least oneof the first forming plate and the second forming plate.

D15. The method of any of clauses D1-D14, wherein separating the firstforming plate and the second forming plate comprises moving only one ofthe first forming plate and the second forming plate.

This summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key features oressential features of the claimed subject matter, nor is it intended tobe used as an aid in determining the scope of the claimed subjectmatter.

Additional features and advantages will be set forth in the descriptionthat follows, and in part will be obvious from the description, or maybe learned by the practice of the invention. The features and advantagesmay be realized and obtained by means of the instruments andcombinations particularly pointed out in the appended claims. These andother features will become more fully apparent from the followingdescription and appended claims, or may be learned by the practice ofthe invention as set forth hereinafter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a flexible pouch useful for self-contained PCR.

FIG. 2 is an exploded perspective view of an instrument for use with thepouch of FIG. 1 , including the pouch of FIG. 1 .

FIG. 3 shows the pouch of FIG. 1 along with bladder components of theinstrument of FIG. 2 .

FIG. 4 shows a motor used in one illustrative embodiment of theinstrument of FIG. 2 .

FIG. 5A shows another embodiment of a pouch, illustrating the pouch inan uncompleted state.

FIG. 5B shows another view of the pouch of FIG. 5A, illustrating thepouch in a completed state.

FIG. 6 shows a separated view of a forming die used during manufactureof the pouch shown in FIGS. 5A and 5B.

FIG. 7 is a separated perspective of the forming die.

FIG. 8 is a front elevation of a first forming plate of the forming dieof FIG. 6 .

FIG. 9 is a front elevation of a second forming plate of the forming dieof FIG. 6 .

FIG. 10 is a cross sectional perspective of the first forming platetaken along line 10-10 of FIG. 8 .

FIG. 11 is a cross sectional perspective of the first forming platetaken along line 11-11 of FIG. 8 .

FIG. 12 is a cross section of the first and second forming platespressed together.

FIG. 13 is a schematic cross-sectional illustration of polymeric sheetspressed between the forming plates to form the pouch.

FIG. 14 is a schematic cross-sectional illustration of a compressedfluid being propelled between the polymeric sheets of FIG. 13 to formchambers in the pouch.

DETAILED DESCRIPTION

Example embodiments are described below with reference to theaccompanying drawings. Many different forms and embodiments are possiblewithout deviating from the spirit and teachings of this disclosure andso the disclosure should not be construed as limited to the exampleembodiments set forth herein. Rather, these example embodiments areprovided so that this disclosure will be thorough and complete, and willconvey the scope of the disclosure to those skilled in the art. In thedrawings, the sizes and relative sizes of layers and regions may beexaggerated for clarity. Like reference numbers refer to like elementsthroughout the description.

Unless defined otherwise, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which the present disclosure pertains.It will be further understood that terms, such as those defined incommonly used dictionaries, should be interpreted as having a meaningthat is consistent with their meaning in the context of the presentapplication and relevant art and should not be interpreted in anidealized or overly formal sense unless expressly so defined herein. Theterminology used in the description of the invention herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting of the invention. While a number of methods and materialssimilar or equivalent to those described herein can be used in thepractice of the present disclosure, only certain exemplary materials andmethods are described herein.

All publications, patent applications, patents or other referencesmentioned herein are incorporated by reference in their entirety. Incase of a conflict in terminology, the present specification iscontrolling.

Various aspects of the present disclosure, including devices, systems,methods, etc., may be illustrated with reference to one or moreexemplary implementations. As used herein, the terms “exemplary” and“illustrative” mean “serving as an example, instance, or illustration,”and should not necessarily be construed as preferred or advantageousover other implementations disclosed herein. In addition, reference toan “implementation” or “embodiment” of the present disclosure orinvention includes a specific reference to one or more embodimentsthereof, and vice versa, and is intended to provide illustrativeexamples without limiting the scope of the invention, which is indicatedby the appended claims rather than by the following description.

It will be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “a tile” includes one, two, or more tiles. Similarly,reference to a plurality of referents should be interpreted ascomprising a single referent and/or a plurality of referents unless thecontent and/or context clearly dictate otherwise. Thus, reference to“tiles” does not necessarily require a plurality of such tiles. Instead,it will be appreciated that independent of conjugation; one or moretiles are contemplated herein.

As used throughout this application the words “can” and “may” are usedin a permissive sense (i.e., meaning having the potential to), ratherthan the mandatory sense (i.e., meaning must). Additionally, the terms“including,” “having,” “involving,” “containing,” “characterized by,”variants thereof (e.g., “includes,” “has,” “involves,” “contains,”etc.), and similar terms as used herein, including the claims, shall beinclusive and/or open-ended, shall have the same meaning as the word“comprising” and variants thereof (e.g., “comprise” and “comprises”),and do not exclude additional, un-recited elements or method steps,illustratively.

As used herein, directional and/or arbitrary terms, such as “top,”“bottom,” “left,” “right,” “up,” “down,” “upper,” “lower,” “inner,”“outer,” “internal,” “external,” “interior,” “exterior,” “proximal,”“distal,” “forward,” “reverse,” and the like can be used solely toindicate relative directions and/or orientations and may not beotherwise intended to limit the scope of the disclosure, including thespecification, invention, and/or claims.

It will be understood that when an element is referred to as being“coupled,” “connected,” or “responsive” to, or “on,” another element, itcan be directly coupled, connected, or responsive to, or on, the otherelement, or intervening elements may also be present. In contrast, whenan element is referred to as being “directly coupled,” “directlyconnected,” or “directly responsive” to, or “directly on,” anotherelement, there are no intervening elements present.

Example embodiments of the present inventive concepts are describedherein with reference to cross-sectional illustrations that areschematic illustrations of idealized embodiments (and intermediatestructures) of example embodiments. As such, variations from the shapesof the illustrations as a result, for example, of manufacturingtechniques and/or tolerances, are to be expected. Thus, exampleembodiments of the present inventive concepts should not be construed aslimited to the particular shapes of regions illustrated herein but areto include deviations in shapes that result, for example, frommanufacturing. Accordingly, the regions illustrated in the figures areschematic in nature and their shapes are not intended to illustrate theactual shape of a region of a device and are not intended to limit thescope of example embodiments.

It will be understood that although the terms “first,” “second,” etc.may be used herein to describe various elements, these elements shouldnot be limited by these terms. These terms are only used to distinguishone element from another. Thus, a “first” element could be termed a“second” element without departing from the teachings of the presentembodiments.

It is also understood that various implementations described herein canbe utilized in combination with any other implementation described ordisclosed, without departing from the scope of the present disclosure.Therefore, products, members, elements, devices, apparatuses, systems,methods, processes, compositions, and/or kits according to certainimplementations of the present disclosure can include, incorporate, orotherwise comprise properties, features, components, members, elements,steps, and/or the like described in other implementations (includingsystems, methods, apparatus, and/or the like) disclosed herein withoutdeparting from the scope of the present disclosure. Thus, reference to aspecific feature in relation to one implementation should not beconstrued as being limited to applications only within thatimplementation.

The headings used herein are for organizational purposes only and arenot meant to be used to limit the scope of the description or theclaims. To facilitate understanding, like reference numerals have beenused, where possible, to designate like elements common to the figures.Furthermore, where possible, like numbering of elements have been usedin various figures. Furthermore, alternative configurations of aparticular element may each include separate letters appended to theelement number.

The term “about” is used herein to mean approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 5%. When such a range is expressed,another embodiment includes from the one particular value and/or to theother particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another embodiment. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint.

The word “or” as used herein means any one member of a particular listand also includes any combination of members of that list.

By “sample” is meant an animal; a tissue or organ from an animal,including, but not limited to, a human animal; a cell (either within asubject (e.g., a human or non-human animal), taken directly from asubject, or a cell maintained in culture or from a cultured cell line);a cell lysate (or lysate fraction) or cell extract; a solutioncontaining one or more molecules derived from a cell, cellular material,or viral material (e.g. a polypeptide or nucleic acid); or a solutioncontaining a non-naturally occurring nucleic acid, which is assayed asdescribed herein. A sample may also be any body fluid or excretion (forexample, but not limited to, blood, urine, stool, saliva, tears, bile,or cerebrospinal fluid) that may or may not contain host or pathogencells, cell components, or nucleic acids. Samples may also includeenvironmental samples such as, but not limited to, soil, water (freshwater, waste water, etc.), air monitoring system samples (e.g., materialcaptured in an air filter medium), surface swabs, and vectors (e.g.,mosquitos, ticks, fleas, etc.).

The phrase “nucleic acid” as used herein refers to a naturally occurringor synthetic oligonucleotide or polynucleotide, whether DNA or RNA orDNA-RNA hybrid, single-stranded or double-stranded, sense or antisense,which is capable of hybridization to a complementary nucleic acid byWatson-Crick base-pairing. Nucleic acids of the invention can alsoinclude nucleotide analogs (e.g., BrdU), and non-phosphodiesterinternucleoside linkages (e.g., peptide nucleic acid (PNA) orthiodiester linkages). In particular, nucleic acids can include, withoutlimitation, DNA, RNA, mRNA, rRNA, cDNA, gDNA, ssDNA, dsDNA, or anycombination thereof.

By “probe,” “primer,” or “oligonucleotide” is meant a single-strandednucleic acid molecule of defined sequence that can base-pair to a secondnucleic acid molecule that contains a complementary sequence (the“target”). The stability of the resulting hybrid depends upon thelength, GC content, and the extent of the base-pairing that occurs. Theextent of base-pairing is affected by parameters such as the degree ofcomplementarity between the probe and target molecules and the degree ofstringency of the hybridization conditions. The degree of hybridizationstringency is affected by parameters such as temperature, saltconcentration, and the concentration of organic molecules such asformamide, and is determined by methods known to one skilled in the art.Probes, primers, and oligonucleotides may be detectably-labeled, eitherradioactively, fluorescently, or non-radioactively, by methodswell-known to those skilled in the art. dsDNA binding dyes may be usedto detect dsDNA. It is understood that a “primer” is specificallyconfigured to be extended by a polymerase, whereas a “probe” or“oligonucleotide” may or may not be so configured.

By “dsDNA binding dyes” is meant dyes that fluoresce differentially whenbound to double-stranded DNA than when bound to single-stranded DNA orfree in solution, usually by fluorescing more strongly. While referenceis made to dsDNA binding dyes, it is understood that any suitable dyemay be used herein, with some non-limiting illustrative dyes describedin U.S. Pat. No. 7,387,887, herein incorporated by reference. Othersignal producing substances may be used for detecting nucleic acidamplification and melting, illustratively enzymes, antibodies, etc., asare known in the art.

By “specifically hybridizes” is meant that a probe, primer, oroligonucleotide recognizes and physically interacts (that is,base-pairs) with a substantially complementary nucleic acid (forexample, a sample nucleic acid) under high stringency conditions, anddoes not substantially base pair with other nucleic acids.

By “high stringency conditions” is meant typically to occur at about amelting temperature (Tm) minus 5° C. (i.e. 5° below the Tm of theprobe). Functionally, high stringency conditions are used to identifynucleic acid sequences having at least 80% sequence identity.

By “lysis particles” is meant various particles or beads for the lysisof cells, viruses, spores, and other material that may be present in asample. Various examples use Zirconium (“Zr”) silicate or ceramic beads,but other lysis particles are known and are within the scope of thisterm, including glass and sand lysis particles. The term “cell lysiscomponent” may include lysis particles, but may also include othercomponents, such as components for chemical lysis, as are known in theart.

While PCR is the amplification method used in the examples herein, it isunderstood that any amplification method that uses a primer may besuitable. Such suitable procedures include polymerase chain reaction(PCR); strand displacement amplification (SDA); nucleic acidsequence-based amplification (NASBA); cascade rolling circleamplification (CRCA), loop-mediated isothermal amplification of DNA(LAMP); isothermal and chimeric primer-initiated amplification ofnucleic acids (ICAN); target based-helicase dependent amplification(HDA); transcription-mediated amplification (TMA), and the like.Therefore, when the term PCR is used, it should be understood to includeother alternative amplification methods. For amplification methodswithout discrete cycles, reaction time may be used where measurementsare made in cycles, doubling time, or crossing point (Cp), andadditional reaction time may be added where additional PCR cycles areadded in the embodiments described herein. It is understood thatprotocols may need to be adjusted accordingly.

While various examples herein reference human targets and humanpathogens, these examples are illustrative only. Methods, kits, anddevices described herein may be used to detect and sequence a widevariety of nucleic acid sequences from a wide variety of samples,including, human, veterinary, industrial, and environmental.

Various embodiments disclosed herein use a self-contained nucleic acidanalysis pouch to assay a sample for the presence of various biologicalsubstances, illustratively antigens and nucleic acid sequences,illustratively in a single closed system. Such systems, includingpouches and instruments for use with the pouches, are disclosed in moredetail in U.S. Pat. Nos. 8,394,608, 8,895,295, 10,464,060, hereinincorporated by reference in their entireties. However, it is understoodthat such pouches are illustrative only, and the nucleic acidpreparation and amplification reactions discussed herein may beperformed in any of a variety of open or closed system sample vessels asare known in the art, including 96-well plates, plates of otherconfigurations, arrays, carousels, and the like, using a variety ofnucleic acid purification and amplification systems, as are known in theart. While the terms “sample well”, “amplification well”, “amplificationcontainer”, or the like are used herein, these terms are meant toencompass wells, tubes, and various other reaction containers, as areused in these amplification systems. In one embodiment, the pouch isused to assay for multiple pathogens. The pouch may include one or moreblisters used as sample wells, illustratively in a closed system.Illustratively, various steps may be performed in the optionallydisposable pouch, including nucleic acid preparation, primary largevolume multiplex PCR, dilution of primary amplification product, andsecondary PCR, culminating with optional real-time detection orpost-amplification analysis such as melting-curve analysis. Further, itis understood that while the various steps may be performed in pouchesof the present invention, one or more of the steps may be omitted forcertain uses, and the pouch configuration may be altered accordingly.While many embodiments herein use a multiplex reaction for thefirst-stage amplification, it is understood that this is illustrativeonly, and that in some embodiments the first-stage amplification may besingleplex. In one illustrative example, the first-stage singleplexamplification targets housekeeping genes, and the second-stageamplification uses differences in housekeeping genes for identification.Thus, while various embodiments discuss first-stage multiplexamplification, it is understood that this is illustrative only.

FIG. 1 shows an illustrative pouch 510 that may be used in variousembodiments, or may be reconfigured for various embodiments. Pouch 510is similar to FIG. 15 of U.S. Pat. No. 8,895,295, with like itemsnumbered the same. Fitment 590 is provided with entry channels 515 athrough 515 l, which also serve as reagent reservoirs or wastereservoirs. Illustratively, reagents may be freeze dried in fitment 590and rehydrated prior to use. Blisters 522, 544, 546, 548, 564, and 566,with their respective channels 514, 538, 543, 552, 553, 562, and 565 aresimilar to blisters of the same number of FIG. 15 of U.S. Pat. No.8,895,295.

Second-stage reaction zone 580 of FIG. 1 is similar to that of U.S. Pat.No. 8,895,295, but the second-stage wells 582 of high density array 581are arranged in a somewhat different pattern. The more circular patternof high density array 581 of FIG. 1 eliminates wells in corners and mayresult in more uniform filling of second-stage wells 582. As shown, thehigh density array 581 is provided with 102 second-stage wells 582.Pouch 510 is suitable for use in the FilmArray® instrument (BioFireDiagnostics, LLC, Salt Lake City, Utah). However, it is understood thatthe pouch embodiment is illustrative only.

While other containers may be used, illustratively, pouch 510 may beformed of two layers of a flexible plastic film or other flexiblematerial such as polyester, polyethylene terephthalate (PET),polycarbonate, polypropylene, polymethylmethacrylate, mixtures,combinations, and layers thereof that can be made by any process knownin the art, including extrusion, plasma deposition, and lamination. Forinstance, each layer can be composed of one or more layers of materialof a single type or more than one type that are laminated together.Metal foils or plastics with aluminum lamination also may be used. Otherbarrier materials are known in the art that can be sealed together toform the blisters and channels. If plastic film is used, the layers maybe bonded together, illustratively by heat sealing. Illustratively, thematerial has low nucleic acid binding and low protein binding capacity.

For embodiments employing fluorescent monitoring, plastic films that areadequately low in absorbance and auto-fluorescence at the operativewavelengths are preferred. Such material could be identified by testingdifferent plastics, different plasticizers, and composite ratios, aswell as different thicknesses of the film. For plastics with aluminum orother foil lamination, the portion of the pouch that is to be read by afluorescence detection device can be left without the foil. For example,if fluorescence is monitored in second-stage wells 582 of thesecond-stage reaction zone 580 of pouch 510, then one or both layers atwells 582 would be left without the foil (e.g., made from opticallytransparent material). In the example of PCR, film laminates composed ofpolyester (Mylar, DuPont, Wilmington Del.) of about 0.0048 inch (0.1219mm) thick and polypropylene films of 0.001-0.003 inch (0.025-0.076 mm)thick perform well. Illustratively, pouch 510 may be made of a clearmaterial capable of transmitting approximately 80%-90% of incidentlight.

In the illustrative embodiment, materials are moved between blisters bythe application of pressure, illustratively pneumatic pressure, upon theblisters and channels. Accordingly, in embodiments employing pressure,the pouch material illustratively is flexible enough to allow thepressure to have the desired effect. The term “flexible” is herein usedto describe a physical characteristic of the material of the pouch. Theterm “flexible” is herein defined as readily deformable by the levels ofpressure used herein without cracking, breaking, crazing, or the like.For example, thin plastic sheets, such as Saran™ wrap and Ziploc® bags,as well as thin metal foil, such as aluminum foil, are flexible.However, only certain regions of the blisters and channels need beflexible, even in embodiments employing pneumatic pressure. Further,only one side of the blisters and channels need to be flexible, as longas the blisters and channels are readily deformable. Other regions ofthe pouch 510 may be made of a rigid material or may be reinforced witha rigid material. Thus, it is understood that when the terms “flexiblepouch” or “flexible sample container” or the like are used, onlyportions of the pouch or sample container need be flexible.

Illustratively, a plastic film may be used for pouch 510. A sheet ofmetal, illustratively aluminum, or other suitable material, may bemilled or otherwise cut, to create a die having a pattern of raisedsurfaces. When fitted into a pneumatic press (illustratively A-5302-PDS,Janesville Tool Inc., Milton Wis.), illustratively regulated at anoperating temperature of 195° C., the pneumatic press works like aprinting press, melting the sealing surfaces of plastic film only wherethe die contacts the film. Likewise, the plastic film(s) used for pouch510 may be cut and welded together using a laser cutting and weldingdevice. Various components, such as PCR primers (illustratively spottedonto the film and dried), antigen binding substrates, magnetic beads,and zirconium silicate beads may be sealed inside various blisters asthe pouch 510 is formed. Reagents for sample processing can be spottedonto the film prior to sealing, either collectively or separately. Inone embodiment, nucleotide tri-phosphates (NTPs) are spotted onto thefilm separately from polymerase and primers, essentially eliminatingactivity of the polymerase until the reaction may be hydrated by anaqueous sample. If the aqueous sample has been heated prior tohydration, this creates the conditions for a true hot-start PCR andreduces or eliminates the need for expensive chemical hot-startcomponents. In another embodiment, components may be provided in powderor pill form and are placed into blisters prior to final sealing.

Pouch 510 may be used in a manner similar to that described in U.S. Pat.No. 8,895,295. In one illustrative embodiment, a 300 μl mixturecomprising the sample to be tested (100 μl) and lysis buffer (200 μl)may be injected into an injection port (not shown) in fitment 590 nearentry channel 515 a, and the sample mixture may be drawn into entrychannel 515 a. Water may also be injected into a second injection port(not shown) of the fitment 590 adjacent entry channel 515 l, and isdistributed via a channel (not shown) provided in fitment 590, therebyhydrating up to eleven different reagents, each of which were previouslyprovided in dry form at entry channels 515 b through 515 l. Illustrativemethods and devices for injecting sample and hydration fluid (e.g. wateror buffer) are disclosed in U.S. Pat. No. 10,464,060, hereinincorporated by reference in its entirety, although it is understoodthat these methods and devices are illustrative only and other ways ofintroducing sample and hydration fluid into pouch 510 are within thescope of this disclosure. These reagents illustratively may includefreeze-dried PCR reagents, DNA extraction reagents, wash solutions,immunoassay reagents, or other chemical entities. Illustratively, thereagents are for nucleic acid extraction, first-stage multiplex PCR,dilution of the multiplex reaction, and preparation of second-stage PCRreagents, as well as control reactions. In the embodiment shown in FIG.1 , all that need be injected is the sample solution in one injectionport and water in the other injection port. After injection, the twoinjection ports may be sealed. For more information on variousconfigurations of pouch 510 and fitment 590, see U.S. Pat. No.8,895,295, already incorporated by reference.

After injection, the sample may be moved from injection channel 515 a tolysis blister 522 via channel 514. Lysis blister 522 is provided withbeads or particles 534, such as ceramic beads or other abrasiveelements, and is configured for vortexing via impaction using rotatingblades or paddles provided within the FilmArray® instrument.Bead-milling, by shaking, vortexing, sonicating, and similar treatmentof the sample in the presence of lysis particles such as zirconiumsilicate (ZS) beads 534, is an effective method to form a lysate. It isunderstood that, as used herein, terms such as “lyse,” “lysing,” and“lysate” are not limited to rupturing cells, but that such terms includedisruption of non-cellular particles, such as viruses. In anotherembodiment, a paddle beater using reciprocating or alternating paddles,such as those described in U.S. Pat. Pub. No. 2019/0344269, hereinincorporated by reference in its entirety, may be used for lysis in thisembodiment, as well as in the other embodiments described herein.

FIG. 4 shows a bead beating motor 819 of instrument 800 of FIG. 2 . Thebead beating motor 819 comprises blades 821 that may be mounted on afirst side 811 of support member 802 of the instrument 800. Blades mayextend through slot 804 to contact pouch 510. It is understood, however,that motor 819 may be mounted on other structures of instrument 800. Inone illustrative embodiment, motor 819 is a Mabuchi RC-280SA-2865 DCMotor (Chiba, Japan), mounted on support member 802. In one illustrativeembodiment, the motor is turned at 5,000 to 25,000 rpm, moreillustratively 10,000 to 20,000 rpm, and still more illustrativelyapproximately 15,000 to 18,000 rpm. For the Mabuchi motor, it has beenfound that 7.2V provides sufficient rpm for lysis. It is understood,however, that the actual speed may be somewhat slower when the blades821 are impacting pouch 510. Other voltages and speeds may be used forlysis depending on the motor and paddles used. Optionally, controlledsmall volumes of air may be provided into the bladder 822 adjacent lysisblister 522. It has been found that in some embodiments, partiallyfilling the adjacent bladder with one or more small volumes of air aidsin positioning and supporting lysis blister during the lysis process.Alternatively, another structure, illustratively a rigid or compliantgasket or other retaining structure around lysis blister 522, can beused to restrain pouch 510 during lysis. It is also understood thatmotor 819 is illustrative only, and other devices may be used formilling, shaking, or vortexing the sample. In some embodiments,chemicals or heat may be used in addition to or instead of mechanicallysis.

Once the sample material has been adequately lysed, the sample is movedto a nucleic acid extraction zone, illustratively through channel 538,blister 544, and channel 543, to blister 546, where the sample is mixedwith a nucleic acid-binding substance, such as silica-coated magneticbeads 533. Alternatively, magnetic beads 533 may be rehydrated,illustratively using fluid provided from one of the entry channel 515c-515 e, and then moved through channel 543 to blister 544, and thenthrough channel 538 to blister 522. The mixture is allowed to incubatefor an appropriate length of time, illustratively approximately 10seconds to 10 minutes. A retractable magnet located within theinstrument adjacent blister 546 (see, e.g., magnet 850, FIG. 2 )captures the magnetic beads 533 from the solution, forming a pelletagainst the interior surface of blister 546. If incubation takes placein blister 522, multiple portions of the solution may need to be movedto blister 546 for capture. The liquid is then moved out of blister 546and back through blister 544 and into blister 522, which is now used asa waste receptacle. One or more wash buffers from one or more ofinjection channels 515 c to 515 e are provided via blister 544 andchannel 543 to blister 546. Optionally, the magnet is retracted and themagnetic beads 533 are washed by moving the beads back and forth fromblisters 544 and 546 via channel 543. Once the magnetic beads 533 arewashed, the magnetic beads 533 are recaptured in blister 546 byactivation of the magnet, and the wash solution is then moved to blister522. This process may be repeated as necessary to wash the lysis bufferand sample debris from the nucleic acid-binding magnetic beads 533.

After washing, elution buffer stored at injection channel 515 f is movedto blister 548, and the magnet is retracted. The solution is cycledbetween blisters 546 and 548 via channel 552, breaking up the pellet ofmagnetic beads 533 in blister 546 and allowing the captured nucleicacids to dissociate from the beads and come into solution. The magnet isonce again activated, capturing the magnetic beads 533 in blister 546,and the eluted nucleic acid solution is moved into blister 548.

First-stage PCR master mix from injection channel 515 g is mixed withthe nucleic acid sample in blister 548. Optionally, the mixture is mixedby forcing the mixture between blisters 548 and 564 via channel 553.After several cycles of mixing, the solution is contained in blister564, where a pellet of first-stage PCR primers is provided, at least oneset of primers for each target, and first-stage multiplex PCR isperformed. If RNA targets are present, a reverse transcription (RT) stepmay be performed prior to or simultaneously with the first-stagemultiplex PCR. First-stage multiplex PCR temperature cycling in theFilmArray® instrument is illustratively performed for 15-20 cycles,although other levels of amplification may be desirable, depending onthe requirements of the specific application. The first-stage PCR mastermix may be any of various master mixes, as are known in the art. In oneillustrative example, the first-stage PCR master mix may be any of thechemistries disclosed in U.S. Pat. No. 9,932,634, herein incorporated byreference, for use with PCR protocols taking 20 seconds or less percycle.

After first-stage PCR has proceeded for the desired number of cycles,the sample may be diluted, illustratively by forcing most of the sampleback into blister 548, leaving only a small amount in blister 564, andadding second-stage PCR master mix from injection channel 515 i.Alternatively, a dilution buffer from 515 i may be moved to blister 566then mixed with the amplified sample in blister 564 by moving the fluidsback and forth between blisters 564 and 566 via channel 562. If desired,dilution may be repeated several times, using dilution buffer frominjection channels 515 j and 515 k, or injection channel 515 k may bereserved, illustratively, for sequencing or for other post-PCR analysis,and then adding second-stage PCR master mix from injection channel 515 hto some or all of the diluted amplified sample. It is understood thatthe level of dilution may be adjusted by altering the number of dilutionsteps or by altering the percentage of the sample discarded prior tomixing with the dilution buffer or second-stage PCR master mixcomprising components for amplification, illustratively a polymerase,dNTPs, and a suitable buffer, although other components may be suitable,particularly for non-PCR amplification methods. If desired, this mixtureof the sample and second-stage PCR master mix may be pre-heated inblister 564 prior to movement to second-stage wells 582 for second-stageamplification. Such preheating may obviate the need for a hot-startcomponent (antibody, chemical, or otherwise) in the second-stage PCRmixture.

In one embodiment, the illustrative second-stage PCR master mix isincomplete, lacking primer pairs, and each of the 102 second-stage wells582 is pre-loaded with a specific PCR primer pair. In other embodiments,the master mix may lack other components (e.g., polymerase, Mg²⁺, etc.)and the lacking components may be pre-loaded in the array. If desired,second-stage PCR master mix may lack other reaction components, andthese components may be pre-loaded in the second-stage wells 582 aswell. Each primer pair may be similar to or identical to a first-stagePCR primer pair or may be nested within the first-stage primer pair.Movement of the sample from blister 564 to the second-stage wells 582completes the PCR reaction mixture. Once high density array 581 isfilled, the individual second-stage reactions are sealed in theirrespective second-stage blisters by any number of means, as is known inthe art. Illustrative ways of filling and sealing the high density array581 without cross-contamination are discussed in U.S. Pat. No.8,895,295, already incorporated by reference. Illustratively, thevarious reactions in wells 582 of high density array 581 aresimultaneously or individually thermal cycled, illustratively with oneor more Peltier devices, although other means for thermal cycling areknown in the art.

In certain embodiments, second-stage PCR master mix contains the dsDNAbinding dye LCGreen® Plus (BioFire Diagnostics, LLC) to generate asignal indicative of amplification. However, it is understood that thisdye is illustrative only, and that other signals may be used, includingother dsDNA binding dyes and probes that are labeled fluorescently,radioactively, chemiluminescently, enzymatically, or the like, as areknown in the art. Alternatively, wells 582 of array 581 may be providedwithout a signal, with results reported through subsequent processing.

When pneumatic pressure is used to move materials within pouch 510, inone embodiment, a “bladder” may be employed. The bladder assembly 810, aportion of which is shown in FIGS. 2-3 , includes a bladder plate 824housing a plurality of inflatable bladders 822, 844, 846, 848, 864, and866, each of which may be individually inflatable, illustratively by acompressed gas source. Because the bladder assembly 810 may be subjectedto compressed gas and used multiple times, the bladder assembly 810 maybe made from tougher or thicker material than the pouch. Alternatively,bladders 822, 844, 846, 848, 864, and 866 may be formed from a series ofplates fastened together with gaskets, seals, valves, and pistons. Otherarrangements are within the scope of this invention. Alternatively, anarray of mechanical actuators and seals may be used to seal channels anddirect movement of fluids between blisters. A system of mechanical sealsand actuators that may be adapted for the instruments described hereinis described in detail in U.S. Pat. Pub. No. 2019/0344269, the entiretyof which is already incorporated by reference.

Success of the secondary PCR reactions is dependent upon templategenerated by the multiplex first-stage reaction. Typically, PCR isperformed using DNA of high purity. Methods such as phenol extraction orcommercial DNA extraction kits provide DNA of high purity. Samplesprocessed through the pouch 510 may require accommodations be made tocompensate for a less pure preparation. PCR may be inhibited bycomponents of biological samples, which is a potential obstacle.Illustratively, hot-start PCR, higher concentration of Taq polymeraseenzyme, adjustments in MgCl₂ concentration, adjustments in primerconcentration, addition of engineered enzymes that are resistant toinhibitors, and addition of adjuvants (such as DMSO, TMSO, or glycerol)optionally may be used to compensate for lower nucleic acid purity.While purity issues are likely to be more of a concern with first-stageamplification, it is understood that similar adjustments may be providedin the second-stage amplification as well.

When pouch 510 is placed within the instrument 800, the bladder assembly810 is pressed against one face of the pouch 510, so that if aparticular bladder is inflated, the pressure will force the liquid outof the corresponding blister in the pouch 510. In addition to bladderscorresponding to many of the blisters of pouch 510, the bladder assembly810 may have additional pneumatic actuators, such as bladders orpneumatically-driven pistons, corresponding to various channels of pouch510. FIGS. 2-3 show an illustrative plurality of pistons or hard seals838, 843, 852, 853, and 865 that correspond to channels 538, 543, 553,and 565 of pouch 510, as well as seals 871, 872, 873, 874 that minimizebackflow into fitment 590. When activated, hard seals 838, 843, 852,853, and 865 form pinch valves to pinch off and close the correspondingchannels. To confine liquid within a particular blister of pouch 510,the hard seals are activated over the channels leading to and from theblister, such that the actuators function as pinch valves to pinch thechannels shut. Illustratively, to mix two volumes of liquid in differentblisters, the pinch valve actuator sealing the connecting channel isactivated, and the pneumatic bladders over the blisters are alternatelypressurized, forcing the liquid back and forth through the channelconnecting the blisters to mix the liquid therein. The pinch valveactuators may be of various shapes and sizes and may be configured topinch off more than one channel at a time. While pneumatic actuators arediscussed herein, it is understood that other ways of providing pressureto the pouch are contemplated, including various electromechanicalactuators such as linear stepper motors, motor-driven cams, rigidpaddles driven by pneumatic, hydraulic or electromagnetic forces,rollers, rocker-arms, and in some cases, cocked springs. In addition,there are a variety of methods of reversibly or irreversibly closingchannels in addition to applying pressure normal to the axis of thechannel. These include kinking the bag across the channel, heat-sealing,rolling an actuator, and a variety of physical valves sealed into thechannel such as butterfly valves and ball valves. Additionally, smallPeltier devices or other temperature regulators may be placed adjacentthe channels and set at a temperature sufficient to freeze the fluid,effectively forming a seal. Also, while the design of FIG. 1 is adaptedfor an automated instrument featuring actuator elements positioned overeach of the blisters and channels, it is also contemplated that theactuators could remain stationary, and the pouch 510 could betransitioned such that a small number of actuators could be used forseveral of the processing stations including sample disruption,nucleic-acid capture, first and second-stage PCR, and processingstations for other applications of the pouch 510 such as immuno-assayand immuno-PCR. Rollers acting on channels and blisters could proveparticularly useful in a configuration in which the pouch 510 istranslated between stations. Thus, while pneumatic actuators are used inthe presently disclosed embodiments, when the term “pneumatic actuator”is used herein, it is understood that other actuators and other ways ofproviding pressure may be used, depending on the configuration of thepouch and the instrument.

Turning back to FIG. 2 , each pneumatic actuator is connected tocompressed air source 895 via valves 899. While only several hoses 878are shown in FIG. 2 , it is understood that each pneumatic fitting isconnected via a hose 878 to the compressed gas source 895. Compressedgas source 895 may be a compressor, or, alternatively, compressed gassource 895 may be a compressed gas cylinder, such as a carbon dioxidecylinder. Compressed gas cylinders are particularly useful ifportability is desired. Other sources of compressed gas are within thescope of this invention. Similar pneumatic control may be provided, forexample, for control of fluid movement in the pouches described herein,or other actuators, servos, or the like may be provided.

Several other components of instrument 810 are also connected tocompressed gas source 895. A magnet 850, which is mounted on a secondside 814 of support member 802, is illustratively deployed and retractedusing gas from compressed gas source 895 via hose 878, although othermethods of moving magnet 850 are known in the art. Magnet 850 sits inrecess 851 in support member 802. It is understood that recess 851 canbe a passageway through support member 802, so that magnet 850 cancontact blister 546 of pouch 510.

However, depending on the material of support member 802, it isunderstood that recess 851 need not extend all the way through supportmember 802, as long as when magnet 850 is deployed, magnet 850 is closeenough to provide a sufficient magnetic field at blister 546, and whenmagnet 850 is fully retracted, magnet 850 does not significantly affectany magnetic beads 533 present in blister 546. While reference is madeto retracting magnet 850, it is understood that an electromagnet may beused and the electromagnet may be activated and inactivated bycontrolling flow of electricity through the electromagnet. Thus, whilethis specification discusses withdrawing or retracting the magnet, it isunderstood that these terms are broad enough to incorporate other waysof withdrawing the magnetic field. It is understood that the pneumaticconnections may be pneumatic hoses or pneumatic air manifolds, thusreducing the number of hoses or valves required. It is understood thatsimilar magnets and methods for activating the magnets may be used inother embodiments.

The various pneumatic pistons 868 of pneumatic piston array 869 are alsoconnected to compressed gas source 895 via hoses 878. While only twohoses 878 are shown connecting pneumatic pistons 868 to compressed gassource 895, it is understood that each of the pneumatic pistons 868 areconnected to compressed gas source 895. Twelve pneumatic pistons 868 areshown, although other configurations are within the scope of the presentinvention.

A pair of temperature control elements are mounted on a second side 814of support 802. As used herein, the term “temperature control element”refers to a device that adds heat to or removes heat from a sample.Illustrative examples of a temperature control element include, but arenot limited to, heaters, coolers, Peltier devices, resistive heaters,induction heaters, electromagnetic heaters, thin film heaters, printedelement heaters, positive temperature coefficient heaters, andcombinations thereof. A temperature control element may include multipleheaters, coolers, Peltiers, etc. In one aspect, a given temperaturecontrol element may include more than one type of heater or cooler. Forinstance, an illustrative example of a temperature control element mayinclude a Peltier device with a separate resistive heater applied to thetop and/or the bottom face of the Peltier. While the term “heater” isused throughout the specification, it is understood that othertemperature control elements may be used to adjust the temperature ofthe sample.

As discussed above, first-stage heater 886 may be positioned to heat andcool the contents of blister 564 for first-stage PCR. As seen in FIG. 2, second-stage heater 888 may be positioned to heat and cool thecontents of second-stage blisters 582 of array 581 of pouch 510, forsecond-stage PCR. It is understood, however, that these heaters couldalso be used for other heating purposes, and that other heaters may beincluded, as appropriate for the particular application.

As discussed above, while Peltier devices, which thermocycle between twoor more temperatures, are effective for PCR, it may be desirable in someembodiments to maintain heaters at a constant temperature.Illustratively, this can be used to reduce run time, by eliminating timeneeded to transition the heater temperature beyond the time needed totransition the sample temperature. Also, such an arrangement can improvethe electrical efficiency of the system as it is only necessary tothermally cycle the smaller sample and sample vessel, not the muchlarger (more thermal mass) Peltier devices. For instance, an instrumentmay include multiple heaters (i.e., two or more) at temperatures setfor, for example, annealing, extension, denaturation that are positionedrelative to the pouch to accomplish thermal cycling. Two heaters may besufficient for many applications. In various embodiments, the heaterscan be moved, the pouch can be moved, or fluids can be moved relative tothe heaters to accomplish thermal cycling. Illustratively, the heatersmay be arranged linearly, in a circular arrangement, or the like. Typesof suitable heaters have been discussed above, with reference tofirst-stage PCR.

When fluorescent detection is desired, an optical array 890 may beprovided. As shown in FIG. 2 , optical array 890 includes a light source898, illustratively a filtered LED light source, filtered white light,or laser illumination, and a camera 896. Camera 896 illustratively has aplurality of photodetectors each corresponding to a second-stage well582 in pouch 510. Alternatively, camera 896 may take images that containall of the second-stage wells 582, and the image may be divided intoseparate fields corresponding to each of the second-stage wells 582.Depending on the configuration, optical array 890 may be stationary, oroptical array 890 may be placed on movers attached to one or more motorsand moved to obtain signals from each individual second-stage well 582.It is understood that other arrangements are possible. Some embodimentsfor second-stage heaters provide the heaters on the opposite side ofpouch 510 from that shown in FIG. 2 . Such orientation is illustrativeonly and may be determined by spatial constraints within the instrument.Provided that second-stage reaction zone 580 is provided in an opticallytransparent material, photodetectors and heaters may be on either sideof array 581.

As shown, a computer 894 controls valves 899 of compressed air source895, and thus controls all of the pneumatics of instrument 800. Inaddition, many of the pneumatic systems in the instrument may bereplaced with mechanical actuators, pressure applying means, and thelike in other embodiments. Computer 894 also controls heaters 886 and888, and optical array 890. Each of these components is connectedelectrically, illustratively via cables 891, although other physical orwireless connections are within the scope of this invention. It isunderstood that computer 894 may be housed within instrument 800 or maybe external to instrument 800. Further, computer 894 may includebuilt-in circuit boards that control some or all of the components, andmay also include an external computer, such as a desktop or laptop PC,to receive and display data from the optical array. An interface,illustratively a keyboard interface, may be provided including keys forinputting information and variables such as temperatures, cycle times,etc. Illustratively, a display 892 is also provided. Display 892 may bean LED, LCD, or other such display, for example.

Other instruments known in the art teach PCR within a sealed flexiblecontainer. See, e.g., U.S. Pat. Nos. 6,645,758, 6,780,617, and9,586,208, herein incorporated by reference. However, including the celllysis within the sealed PCR vessel can improve ease of use and safety,particularly if the sample to be tested may contain a biohazard. In theembodiments illustrated herein, the waste from cell lysis, as well asthat from all other steps, remains within the sealed pouch. Still, it isunderstood that the pouch contents could be removed for further testing.

Turning back to FIG. 2 , instrument 800 includes a support member 802that could form a wall of a casing or be mounted within a casing.Instrument 800 may also include a second support member (not shown) thatis optionally movable with respect to support member 802, to allowinsertion and withdrawal of pouch 510. Illustratively, a lid may coverpouch 510 once pouch 510 has been inserted into instrument 800. Inanother embodiment, both support members may be fixed, with pouch 510held into place by other mechanical means or by pneumatic pressure.

In the illustrative example, heaters 886 and 888 are mounted on supportmember 802. However, it is understood that this arrangement isillustrative only and that other arrangements are possible. Illustrativeheaters include Peltiers and other block heaters, resistive heaters,electromagnetic heaters, and thin film heaters, as are known in the art,to thermocycle the contents of blister 864 and second-stage reactionzone 580. Bladder plate 810, with bladders 822, 844, 846, 848, 864, 866,hard seals 838, 843, 852, 853, and seals 871, 872, 873, 874 form bladderassembly 808, which may illustratively be mounted on a moveable supportstructure that may be moved toward pouch 510, such that the pneumaticactuators are placed in contact with pouch 510. When pouch 510 isinserted into instrument 800 and the movable support member is movedtoward support member 802, the various blisters of pouch 510 are in aposition adjacent to the various bladders of bladder assembly 810 andthe various seals of assembly 808, such that activation of the pneumaticactuators may force liquid from one or more of the blisters of pouch 510or may form pinch valves with one or more channels of pouch 510. Therelationship between the blisters and channels of pouch 510 and thebladders and seals of assembly 808 is illustrated in more detail in FIG.3 .

Self-Contained Reaction Vessels and Methods

FIGS. 5A and 5B show another illustrative embodiment of a pouch 100 a,100 b (also referred to herein as a reaction container, a reactionvessel, and/or a science card) that may be used in various embodiments,or may be reconfigured for various embodiments described herein for PCR,microbial testing, immunologic testing, or for a variety of other tests.FIG. 5A shows an example of an uncompleted pouch 100 a and FIG. 5B showsan illustrative example of a completed pouch 100 b with a samplecollection swab 103, various reagents (e.g., lysis buffer 107 and lysisbeads 105, magnetic beads 115, etc.), a second stage reaction array 109,and other components that may be added to pouch 100 b at the time ofmanufacture. Pouch 100 a, 100 b shown in FIGS. 5A and 5B can bemanufactured according to the methods described herein. Pouch 100 a, 100b may be configured for use in an instrument described in U.S. Pat. Pub.No. 2019/0046989, herein incorporated by reference, or in a variety ofother instruments, such as the instrument as described and shown abovewith reference to FIG. 2 .

The illustrative pouch 100 a, 100 b of FIGS. 5A and 5B includes a numberof zones or blisters where sample preparation, nucleic acidamplification, and detection can occur. The illustrative pouch 100 a,100 b may include a sample input chamber 102, a sample preparationchamber 104, a sample wash reagent chamber 106, reactant recovery/washchambers 112 and 114, a first reaction chamber 116, and a secondreaction chamber 108, and one or more liquid reagent blisters 110 a-110g that may be filled with aqueous reagents at the time of manufacture(as shown at pouch 100 b of FIG. 5B). In some embodiments, one or moreof the liquid reagents in, for example, blisters 110 a-110 g may bereplaced with dried reagents that can be rehydrated at the time of useby liquid sample or other liquid reagents. Reagents in chambers andblisters 104, 106, 114, and 110 a-110 g may be added to pouch 100 a, 100b at the time of manufacture through access channels 243 a-243 j (FIG.5A) that are formed during the manufacturing process. Likewise, asecond-stage reaction array 109 may be inserted into pouch 100 a viaopening 244. After reagents are added, these access openings 243 a-243 jand 244 may be sealed (e.g., heat sealed) to seal the reagents andsecond-stage array in pouch 100 b. Because pouch 100 a, 100 b may befabricated from barrier films that have very low rates of water vaporand oxygen transmission, aqueous liquid reagents and/or dried reagentsin pouch 100 b that may be added at the time of manufacture may bestable under ambient storage conditions for many months or a year ormore (e.g., 3 months, 6 months, 1 year, or more). Pouch 100 a, 100 b mayhave any combination and configuration of chambers, blisters, and fluidchannels. Below, adaptable methods for manufacturing an assay devicehaving formed reaction chambers, blisters, channels, and the like fromsheets of film material are described. In one example, the methodsdescribed herein can be used for manufacturing the pouch 100 a, 100 b.It is understood that such methods of manufacture may also be applied todevices having different configurations or different uses, such as anyof the pouches or reaction containers described above or as described inU.S. Pat. Pub. No. 2020/0261914, the entirety of which is incorporatedherein by reference.

The illustrative pouch 100 a, 100 b includes a fluidic circuit (i.e., aninterconnected series of reaction chambers, channels, and the like influid communication) that can be used for fluid movement in the pouch100 a, 100 b. In the illustrated example, the fluidic circuit of pouch100 a, 100 b includes a series of fluidically connected chambers (e.g.,102, 104, 112, 114, 116, 108/109 etc.), blisters (e.g., 106 and 110a-110 g) and channels (122, 125 a, 125 b, 128, 130, 124 a-124 g, and129) illustratively for sample input, cell lysis and nucleic acidrecovery, a first-stage PCR, a second-stage PCR, and detection ofamplification. Channels 122, 125 a, 125 b, 128, 130, 124 a-124 g, and129 may be sealed at the time of manufacture (e.g., by lamination of thepouch films), but such channels can be opened in use (e.g., by forcingfluid through the channels to peel apart the laminated films) to permitfluid movement between blisters and chambers. In an instrument designedto use pouch 100 b for an assay, channel opening may be selectivelycontrolled and opened channels may be reclosed with the use of hardseals that press on the exterior surface of the pouch and pinch off thechannels (e.g., as described above with reference to FIG. 2 ).

The illustrative pouch 100 b includes a sample input chamber 102 thatmay be used for inputting a sample into the pouch 100 b. In oneembodiment, the sample input chamber 102 includes and a swab 103.Illustratively, the swab 103 may be used for collecting a sample (e.g.,from a throat or nasopharyngeal swab site) and then returned with samplethereon back into the sample input chamber 102, but this is illustrativeonly. In other embodiments, a variety of liquid, semi-liquid,semi-solid, and solid sample types may be introduced directly into thesample input chamber 102. For example, a transfer pipette or the likemay be used for introducing a liquid sample (e.g., whole blood, positiveblood culture, or urine) directly into the sample input chamber 102.Alternatively, swab 103 can be used to insert into the pouch 100 b asample previously collected by other means or devices. Sample inputchamber 102 may be fluidically connected to a sample preparation chamber104 via channel 122. Sample preparation chamber 104 may include a lysisbuffer 107 and lysis beads 105 (e.g., zirconium silicate beads) that maybe used for lysis of cells in a sample. In one embodiment, sample may bewashed off of/recovered from swab 103 by flushing lysis buffer 107through channel 122 back and forth between sample input chamber 102 andsample preparation chamber 104. Cells in the sample may be lysed inchamber 104 by agitating (e.g., bead beating) the sample, lysis buffer,and lysis beads in chamber 104 (e.g., such as described above withreference to FIGS. 1-4 ). If sample lysis is not needed, in someembodiments (not shown) the pouch 100 b may include a reconfiguredchannel 122 that bypasses the sample preparation chamber 104 and thatmay be in direct fluid communication with chamber 112. In theillustrated embodiment, sample preparation chamber 104 is fluidicallyconnected to chambers 112, 114, and 102 for, for example, nucleic acidrecovery from the lysed sample and for waste disposal. In theillustrated embodiment, chamber 114 includes a quantity of magneticsilicate beads 115. The lysed sample may be mixed with the magneticsilicate beads 115 for nucleic acid recovery. For other embodiments,chambers 112 and 114 may contain other reagents (e.g., an immunologiccapture reagent) for other types of assays. Following nucleic acidrecovery, the magnetic silicate beads 115 may be recaptured (e.g., inblister 112 or 114) by a magnet (not shown) external to the pouch 100 band then washed with a wash buffer (for example, a wash buffer may beprovided from blister 106). Depending on the assay, one or more washesmay be performed. Following the wash(es), the magnetic beads may berecaptured by the magnet and the recovered nucleic acids may be elutedinto chamber 106 with an elution buffer (for example, an elution buffermay be provided from blister 110 a).

In one embodiment, the eluted nucleic acids in chamber 106 may betransferred to chamber 116 for a first-stage nucleic acid amplificationreaction (e.g., a PCR reaction). Reagents for first-stage PCR may bepre-loaded in pouch 100 b in liquid and/or dried form at the time ofmanufacture and may be introduced into chamber 116 from, for example,one or more of reagent blisters 110 b-110 g. In some embodiments,chamber 116 of pouch 100 b may include dried reagents 118 in addition toor instead of the liquid reagents that may be provided from reagentblisters 110 b-110 g. In one example, some reagents may be more stablein dried form (e.g., reverse transcriptase, PCR primers, etc.) and maybe, as a result, spotted onto the film and dried in spots 120 prior tolamination. In another example, one or more of the liquid reagents maybe more storage-stable in the absence of one or more reactioncomponents. In such cases, the reaction component(s) may be spotted ontoone of the layers of film at the time of manufacture. An illustrativeprocess for spotting, drying, and incorporating such dried reagents intoan assay device will be described below in reference to themanufacturing method(s) described herein. If such spots 120 are present,they may be rehydrated when the sample and/or the liquid reagents areintroduced into chamber 116.

After the first-stage nucleic amplification reaction, a portion of theproduct may be removed (e.g. plunged) from chamber 116 and a dilutionsolution and second-stage nucleic acid amplification reagent (e.g., PCRreagents) may be added to chamber 116 from, for example, one or more ofreagent blisters 110 b-110 g. The mix for the second-stage nucleic acidamplification reaction (e.g., a PCR reaction) may then be introducedinto the wells of card 109 of chamber 108 for the second-stage nucleicacid amplification reaction and detection of amplified targets.Additional discussion of such pouches or reaction containers and theiruses described above may be found in U.S. Pat. Pub. No. 2019/0046989 orU.S. Pat. Pub. No. 2020/0261914, the entireties of which were alreadyincorporated herein by reference.

Illustratively, pouch 100 a, 100 b may be formed of two or more layersof a flexible plastic film or other flexible material such as polyester,polyethylene, polyethylene terephthalate (PET), polycarbonate,polypropylene (PP), polymethylmethacrylate, mixtures, combinations, andlayers thereof that can be made by any process known in the art,including extrusion, plasma deposition, and lamination. For instance,each layer can be composed of one or more layers of material of a singletype or more than one type that are laminated or fused together. Oneoperative example is a bilayer plastic film that includes a PET layerand a PP layer. In one embodiment, flexible polymeric material having athickness in a range of about 0.02 mm to about 0.1 mm is used. Metalfoils or plastics with aluminum lamination also may be used.Illustratively, the material has low nucleic acid binding and lowprotein binding capacity. If plastic film is used, selected portions ofthe film layers may be bonded together, illustratively by heat sealingor laser welding. If fluorescence detection is used, opticallytransparent material may be used in the appropriate areas of the pouch(e.g., in the vicinity of the second-stage array).

In some embodiments, a barrier film may be used in one or more of thelayers used to form the pouch 100 a, 100 b. For instance, barrier filmsmay be desirable for some applications because they have low water vaporand/or oxygen transmission rates that may be lower than conventionalplastic films. Because liquid reagents may be provided in pouch 100 b atthe time of manufacture, the low water vapor and/or oxygen transmissionrates that are associated with barrier films can prevent evaporation ofwater from the reagents and prevent oxidation of reagents between thetime of manufacture and the time of use (e.g., up to three months, up tosix months, up to one year, or more). Similarly, because certain driedreagents may be provided in pouch 100 b at the time of manufacture, thelow water vapor and/or oxygen transmission rates that are associatedwith barrier films can also prevent degradation of these dried reagentsbecause environmental water and oxygen are less able to penetrate thepouch between the time of manufacture and the time of use. In oneexample, typical barrier films may have water vapor transmission rates(WVTR), as measured, for example, according to ASTM F1249, as low as 0g/m²/24 hrs (i.e., the WVTR may be too low to be measured by ASTMF1249), or a WVTR in a range of 0 g/m²/24 hrs to about 3 g/m²/24 hrs(e.g., about 0.01 g/m²/24 hrs to about 3 g/m²/24 hrs), preferably in arange of about 0.05 g/m²/24 hrs to about 2 g/m²/24 hrs (e.g., no morethan about 1 g/m²/24 hrs) and oxygen transmission rates, as measured,for example, according to ASTM D3985, as low as 0 cc/m²/24 hrs (i.e.,the oxygen transmission rate may be too low to be measured by ASTMD3985), or an oxygen transmission rate in a range of 0 cc/m²/24 hrs toabout 2 cc/m²/24 hrs (e.g., about 0.01 cc/m²/24 hrs to about 2 cc/m²/24hrs), preferably in a range of about 0.05 cc/m²/24 hrs to about 2cc/m²/24 hrs (e.g., no more than about 1 cc/m²/24 hrs). Examples ofbarrier films include, but are not limited to, films that can bemetallized by vapor deposition of a metal (e.g., aluminum or anothermetal) or sputter coated with an oxide (e.g., Al₂O₃ or SiO_(x)) oranother chemical composition. A common example of a metallized film isaluminized Mylar, which is metal coated biaxially oriented PET (BoPET).In some applications, coated barrier films can be laminated with a layerof polyethylene, PP, or a similar thermoplastic, which providessealability and improves puncture resistance. As with conventionalplastic films, barrier films layers used to fabricate a pouch may bebonded together, illustratively by heat sealing. Illustratively, thematerial has low nucleic acid binding and low protein binding capacity.Other barrier materials are known in the art that can be sealed togetherto form the blisters and channels.

In one embodiment, a first polymeric sheet 200 (e.g., a layer of aflexible plastic film or other flexible material as described above)includes a first inner planar face 202 and a first outer planar face 204(see, e.g., FIG. 14 ). A second polymeric sheet 210 (e.g., a layer of aflexible plastic film or other flexible material as described above)includes a second inner planar face 212 and a second outer planar face214. Illustratively, at least one of the first polymeric sheet 200 andthe second polymeric sheet 210 can include a barrier film or other watervapor and/or oxygen barrier material. In one embodiment, both the firstpolymeric sheet 200 and the second polymeric sheet 210 include a barrierfilm or other water vapor and/or oxygen barrier material. In oneembodiment, the first and second polymeric sheets 200, 210 are formed asone piece, such that the first inner planar face 202 and the secondinner planar face 212 are contiguous, and the first outer planar face204 and the second outer planar face 214 are contiguous. For example,the first and second polymeric sheets 200, 210 can be formed from onepolymeric sheet that is folded onto itself (e.g., folded in half). Inanother embodiment, the first and second polymeric sheets 200, 210 areseparate polymeric sheets joined together (e.g., laminated together) andreformed according to the methods described herein to form the pouch 100a, 100 b. Illustratively, the first and second polymeric sheets 200 and210 may include a PET outer layer, an aluminized barrier layer or a“clear” barrier layer (e.g., an aluminum oxide or silicon oxide sputtercoated coated barrier) and an inner PP layer. Preferably, the firstpolymeric and second polymeric sheets 200 and 210 may have a WVTR in arange of about 0 g/m²/24 hrs to about 3 g/m²/24 hrs (e.g., 0.1 g/m²/24hrs or less). Typically, aluminized barrier layers offer greater barrierproperties as compared to sputter coated metal oxide “clear” barrierlayers, but both offer significantly greater barrier properties thatsimilar films without barrier layers. Illustratively, the firstpolymeric sheet 200 may include a polyester (e.g., PET) outer layer, analuminized barrier layer, and a PP inner layer. Illustratively, thesecond polymeric sheet 210 may include a polyester (e.g., PET) outerlayer, a sputter coated metal oxide “clear” barrier layer, and a PPinner layer.

In one embodiment, one or more polymeric sheets may be used to form areaction container (e.g., pouch 100 a, 100 b). The polymeric sheetsinclude an inner planar face that forms the inside of the reactioncontainer and an outer planar face that forms the outside surface of thereaction container. In the above examples, the polypropylene (PP) layerforms the inside of the reaction container and the polyester layer formsthe outside of the reaction container. This is only illustrative,however; the materials that form the inside and outside surfaces of thereaction container may be varied depending on the application, but themethods described herein may be adapted for fabrication of a reactioncontainer from any polymeric sheet material.

In one embodiment, one polymeric sheet may be used to form a reactioncontainer (e.g., pouch 100 a, 100 b). For example, a polymeric sheet(e.g., polymeric sheet 200 or polymeric sheet 210) may be folded ontoitself (e.g., folded in half) so that the inner planar faces of the twoparts of the folded sheet contact each other. In another embodiment, thefirst and second polymeric sheets 200 and 210 may be used to form areaction container. In one embodiment, a method of forming a reactioncontainer may include steps of providing the polymeric sheets 200, 210(i.e., one folded sheet or a first polymeric sheet and a secondpolymeric sheet), contacting the inner planar faces of the polymericsheets, pressing the polymeric sheets between a first forming plate anda second forming plate, and propelling a compressed fluid between theinner planar faces of the polymeric sheets while the polymeric sheetsare pressed between the forming plates to reform selected portions ofthe polymeric sheets into one or more shapes defined by the formingplates. In one embodiment, at least one of the first or second formingplates has one or more recesses for forming one or more reactionchambers, fluid flow channels, reagent chambers, or sample chambers inselected portions of the polymeric sheets. That is, when the compressedfluid is forced between the inner planar faces of the polymeric sheets,the polymeric sheets can expand outward into the spaces defined by theforming plates. When the pressure is released, the selected areas of thepolymeric sheets that were expanded into the forming plates retain theshape(s) (e.g., reaction chambers, sample chambers, reagent blisters,etc.) defined by the forming plates.

In some embodiments, the polymeric sheets (i.e., the folded polymericsheet or the first and second polymeric sheets) may be laminated to oneanother (i.e., reversibly sealed to one another) prior to the pressingand propelling steps. For example, the polymeric sheets may be pressedbetween hot plates or between heated rollers to reversibly seal theinner planar faces to one another. In the example described above, theinner polypropylene layers of the sheets may be heated and pressedtogether such that the opposing polypropylene layers are sealed to oneanother, but sealed in such a way that the sheets can be peeled apart(i.e., the sheets are reversibly sealed together). In the specificembodiment where the opposing polypropylene layers are reversibly sealedto one another, the lamination may occur at a temperature in a range ofabout 110° C. to about 130° C. (e.g., 120° C.). In a specificembodiment, the polymeric sheets may be passed between heated rollers ata temperature of about 110° C. to about 130° C. to reversibly seal theinner planar faces of the sheets together. In one example, the heatedrollers exert a pressure of approximately 10 PSI to 100 PSI (˜0.07 MPato ˜0.7 MPa) (e.g., about 40-50 PSI) and the sheets are exposed to thetemperature of about 110° C. to about 130° C. for approximately 0.05 to0.5 seconds (e.g., 0.1 seconds). In some embodiments, thetime/temperature/pressure parameters may be adjusted for different filmmaterials and/or to create reversible seals having different peelstrengths.

As discussed herein above, pouch 100 b may include dried reagent spots120. In one embodiment, liquid reagents may be spotted onto at least oneof the first and second polymeric sheets 200, 210 and then dried (e.g.,air dried) prior to laminating the first and second polymeric sheets. Inone embodiment, liquid reagents may be added dropwise onto at least oneof the first and second polymeric sheets 200, 210 and the liquid may besubsequently air dried. The liquid may be spotted onto the film manuallyor with the aid of a liquid handling robot. In one embodiment, theliquid reagents may be spotted onto the films with a modified ink jetprinter head. In one embodiment, the spotted reagents may beelectrostatically transferred to the film layer as dried powder in aprocess similar to laser printing or photocopying. This would eliminatethe need to dry the reagent prior to lamination.

In some embodiments, one or more seal lines may be formed on thepolymeric sheets prior to the pressing and propelling steps. In oneembodiment, the one or more seal lines define boundaries of a fluidiccircuit (i.e., an interconnected flow path) that can be used for fluidmovement in the reaction container (e.g., pouch 100 a, 100 b). In oneembodiment, the fluidic circuit may include one or more reactionchambers, fluid flow channels, or sample chambers (e.g., as describedabove with reference to FIGS. 5A and 5B). In one embodiment, the one ormore seal lines may be formed by a heat-sealing apparatus, alaser-welding apparatus, a sonic welding apparatus, or other apparatusesknown in the art. Suitable examples of heat sealing apparatuses include,but are not limited to, heated plates that have lines that define thefluidic circuit formed thereon. For example, metal plates with raised,hot portions tracing the fluidic circuit may serve to permanently sealthe polymeric sheets to one another in select areas to define theboundaries of the fluidic circuit. In one embodiment, such plates mayhave cooler regions between the seal lines (cooler regions may befilled, for example, with syntactic foam or the like) such that only theseal lines are sealed. Likewise, examples of laser welding apparatusesare well known in the art. An example of a laser welding apparatus mayinclude a laser with a wavelength chosen to weld the polymeric sheetstogether to create permanent seal lines and a computer controller thatcan be programmed to control the path of the laser. In one embodiment,the first and second polymeric sheets may be laminated to one anotherand then one or more seal lines may be formed to define the fluidiccircuit prior to the pressing and propelling steps. In some embodiments,boundaries of the seal lines may correspond to the shapes in the formingplates such that the seal lines and the forming plates define thereformed shapes when the compressed fluid is propelled between thepolymeric sheets. The polymeric sheets that include openings formed bythe compressed fluid and the forming plates can be removed from theforming plates, and the manufacture of the reaction container can becompleted by inserting reagents (e.g., lysis components, wash reagents,PCR reagents, etc.) into the correct blisters and chambers that havebeen formed.

Referring to FIGS. 6 and 7 , a forming die 218 includes a first formingplate 220 and a second forming plate 230. The first and second formingplates 220, 230 of the forming die 218 are configured and dimensioned tobe pressed or clamped together to form the polymeric sheets 200, 210into a reaction container (e.g., pouch 100 a, 100 b). The first formingplate 220 includes one or more recesses 222. The second forming plate230 includes one or more recesses 232. Illustratively, recesses 222 aresubstantially aligned with recesses 232 when the first and secondforming plates 220, 230 are pressed together so as to create formingchambers. For example, as shown in FIGS. 8-9 , recesses 222 aresubstantially a mirror image of and aligned with recesses 232. As shownin FIG. 13 , illustrative recess 222 a is substantially a mirror imageof and aligned with illustrative recess 232 a such that when the firstand second forming plates are pressed together a forming chamber 234 isdefined. The recesses 222, 232 are shaped so as to reform portions ofthe first and second polymeric sheets 200, 210 into desired shapes toform the applicable chambers (e.g., reagent reservoirs, samplepreparation chambers, etc.). Other configurations are within the scopeof the present invention, such as recesses that create asymmetricalforming chambers. For instance, while recesses 222 a and 232 a of FIG.13 are shown as substantial mirror images, they may have differentshapes or different depths in some embodiments. For example, it may bepreferable in some embodiments to reform one type of polymeric sheetmore than another. In an illustrative example, it may be preferable toreform one barrier film more than another because one barrier may bemore or less susceptible to degradation of barrier properties as aresult of the reforming process described herein. In anotherillustrative example, it may be preferable to reform one film materialmore than another because the films have different stretchproperties—e.g., different abilities to be stretched and reformed. Assuch, in one illustrative example, only one forming plate may includerecesses such that only one of the film materials is reformed. Otherconfigurations are within the scope of the present invention.

The forming chambers defined between the first and second forming plates220, 230 (e.g., such as forming chamber 234 defined by recesses 222 aand 232 a, illustrated in FIG. 13 ) are arranged to form the blisters,chambers, and/or channels in a reaction container (e.g., pouch 100 a,100 b). For example, in the illustrated embodiment as best seen in FIGS.8-9 , recesses 222 b, 232 b align to create a forming chamber for thesample input chamber 102. Recesses 222 c, 232 c align to create aforming chamber for the sample preparation chamber 104. Recesses 222 d,232 d align to create a forming chamber for the sample reagent washchamber 106. Recesses 222 e, 232 e align to create a forming chamber forreaction chamber 108. Recesses 222 f, 232 f align to create a formingchamber for reagent blisters 110. In the illustrated embodiment, each ofthe recesses 222, 232 includes at least one vent hole 236 for ventingair expelled from the respective recess during forming of the reactioncontainer, as will be described in further detail below. Otherconfigurations are within the scope of the invention, such as differentrecess and forming chambers that may be required depending on theconfiguration of the reaction container or pouch.

In one embodiment, the first inner planar face 202 and the second innerplanar face 212 of the film layers 200 and 210 are contacted and pressedbetween the first forming plate 220 and the second forming plate 230.Subsequent to pressing film layers 200 and 210 between forming plates220 and 230, a compressed fluid (e.g., compressed gas, compressed air,compressed liquid, or other suitable compressed fluid) may be propelledbetween the first and second inner planar faces 202, 212. The compressedfluid forces portions of the first and second polymeric sheets 200, 210into the respective recesses 222, 232, thereby forming an opening orhollow area 240 between the first and second polymeric sheets (forexample, reforming the polymeric sheets to include the opening or hollowarea between the sheets) (see, e.g., FIGS. 13 and 14 ). This opening orhollow area 240 illustratively can be a blister, a chamber, and/or achannel in the pouch 100 a, 100 b. If the polymer sheets have beenlaminated prior to pressing the film layers between the forming plates,the compressed fluid peels the sheets apart in the selected portions toform the hollow area according to the structure of the forming plates,as described in further detail below. Alternatively, the films may notbe laminated together prior to pressing and reforming. As the compressedfluid forces portions of the polymeric sheets 200, 210 into the recesses222, 232, any air that is displaced from the recesses can exit theforming plates 220, 230 through the at least one vent hole 236 in eachrecess. Each vent hole 236 vents to outside the respective forming plate220, 230 (see, e.g., FIG. 10 ).

In one embodiment, as shown in FIGS. 5A, 13, and 14 , one or more holes260 may be cut in one of the first and second polymeric sheets 200, 210.In the illustrated embodiment, holes 260 are cut in the first polymericsheet 200 but not in the second polymeric sheet 210, although thereverse or having some holes in each of the sheets are possible andother configurations are within the scope of the present invention. Theholes 260 permit fluid communication for a compressed fluid source tobetween the first and second polymeric sheets 200, 210. As best seen inFIGS. 10-11 , the forming die 218 includes one or more conduits 262 influid communication with the holes 260 (see, e.g., FIG. 11 ). Asillustrated in FIG. 13 , polymeric sheets 200 and 210 may be compressedbetween plates 220 and 230 such that hole(s) 260 are aligned withconduit 262.

As shown in FIG. 14 , compressed fluid (illustrated by the arrows) froma compressed fluid source (not shown) enters the forming die 218 andtravels through a conduit 262 to a respective hole 260 in the firstpolymeric sheet 200 to be propelled between the first polymeric sheetand the second polymeric sheet 210. In the illustrated embodiment, thefirst polymeric sheet 200 includes a hole 260 corresponding to eachopening or hollow area 240 that must be formed in the pouch 100 a, 100 b(e.g., there is a hole 260 corresponding to each blister, chamber,and/or channel that will be formed by the compressed fluid and theforming die). The forming die 218 includes conduits 262 in fluidcommunication with each hole 260 so that compressed fluid can bepropelled into each hole and between the first and second polymericsheets 200, 210 to each location where an opening 240 must be formed(e.g., to opening 240 from hole 260). As shown in FIGS. 13 and 14 , theforming plates 220 and 230 may include shallow cut-out regions 223 a and233 a adjacent to conduit 262 such that the film layers 200 and 210 canexpand adjacent to conduit 262, as shown at 223 and 233, so that thecompressed fluid can flow between the layers and expand the film to formopening 240. Illustratively, the conduit and hole configuration permitsall of the openings to be formed simultaneously or substantiallysimultaneously. The channels connecting the holes 260 to the reformedopenings 240 may subsequently be used to inject fluids (e.g., reagents)into the reaction container (e.g., pouch 100 a, 100 b). For example, thearea that was expanded around 223 and 233 may form an access channel 243that can be used to inject fluids (e.g., reagents) into opening 240.

Illustratively, the first and second polymeric sheets 200, 210 may belaminated together prior to being pressed or clamped between the formingplates 220, 230. Illustratively, the lamination may reversibly adherethe sheets 200, 210 together, allowing for ease of handling but stillallowing for formation of the blisters, chamber, and channelstherebetween. In embodiments where the films are laminated, thecompressed fluid can also peel apart the lamination so that thecompressed fluid can flow between layers 200, 210 and expand the film toform opening 240. Furthermore, the first and second polymeric sheets200, 210 may be sealed at certain locations prior to being pressed orclamped between the forming plates 220, 230. For example, one or moreseal lines may be made to join the first and second polymeric sheets200, 210 to define each opening or hollow area 240 that will be formedbetween the sheets. As shown in cross section in FIGS. 13 and 14 , filmlayers 200 and 210 may include a seal line 242 (e.g., a laser weld line)that joins film layers 200 and 210 adjacent to the opening defined byrecesses 222 a and 232 a. In the illustrated cross section, only oneedge of the seal line 242 is visible but, as explained elsewhere herein,the seal line may trace around and define the boundaries of opening 240.In one embodiment, film layers 200 and 210 may be laminated to oneanother prior to forming heat seals (e.g., heat seal 242) and prior toclamping the film layers 200 and 210 between plates 220 and 230.Suitably, the seal lines may be formed by laser welding, heat sealing,sonic welding, or other suitable sealing method. Preferably, the seallines generally align with respective recess pairs 222, 232 of theforming plates 220, 230 when the sheets 200, 210 are pressed between theplates.

In one embodiment, a laminated polymeric sheet comprising first andsecond polymeric sheets 200, 210 may be joined with the one or more seallines (e.g., laser welds) to join the first and second polymeric sheetsand define a configuration of blisters, chambers, and/or channels (i.e.,a fluidic circuit). Holes 260 may be cut in the first and/or secondpolymeric sheets 200, 210 during the same laser welding operation or ata different time. In addition, one or more alignment holes (130 and 131,FIG. 5B) for aligning the sheets and laser weld lines in the formingplates may be cut in the first and/or second polymeric sheets 200, 210during the same laser welding operation or at a different time. Thelaminated and laser-welded polymeric sheets 200, 210 may then be pressedor clamped in the forming die 218 such that the holes 260 (which arealso pressed in the forming die) are each in fluid communication withrespective conduits 262. In addition, the seal lines joining the firstand second polymeric sheets 200, 210 may generally align with respectiverecesses 222, 232 in the first and second forming plates 220, 230 of theforming die 218. A compressed fluid is then propelled through theconduits 262, into the respective holes 260, and between the first andsecond polymeric sheets 200, 210. The compressed fluid is propelleddirectly on the inner planar surfaces 202, 212 of the first and secondpolymeric sheets 200, 210. The compressed fluid expands selected areasof the first and second polymeric sheets 200, 210 into channel formers223 a and 233 a and forming chambers 234 formed by aligned recess pairs222, 232 to form openings 240 between the first and second polymericsheets.

Displaced air is vented from the recesses 222, 232 through respectiveventing holes 236. The openings 240 generally correspond to the seallines previously made to join the first and second polymeric sheetstogether. In one embodiment, films 200 and 210 may be expanded intoforming chambers 234 at a constant pressure. In another embodiment,films 200 and 210 may be expanded into forming chambers 234 at avariable pressure. For example, film expansion may be started at aninitial pressure of about 10 PSI (˜0.07 MPa) and them ramp up linearlyover about 1-3 seconds to a pressure of about 100-160 PSI (˜0.7 MPa-1.1MPa); the pressure may be held at the higher pressure for 1-3 seconds.In one embodiment, the pressure may be regulated with a rampingregulator or a solenoid with a small opening that acts as a pressurerestrictor. Other variable pressure protocols and devices are within thescope of the present invention.

Illustratively, the polymeric sheets 200, 210 are cold formed in theforming die 218 to create the openings 240. The polymeric sheets 200,210, which now include openings formed by the compressed fluid and theforming plates 220, 230, can be removed from the forming plates, and themanufacture of the pouch 100 a, 100 b can be completed by inserting aselection of one or more liquid and/or dried reagents, wash solutions,etc. into the correct blisters and chambers that have been formed.

Although the above-described method does not include heating thepolymeric sheets 200, 210 prior to forming the sheets between theforming plates 220, 230, it is understood that the sheets may be heatedto a softening temperature (e.g., a plastic transition temperature)prior to or during this process to aid formation of the desired shapes.For example, the sheets 200, 210 may be pressed between heating platesfor heating prior to being pressed or clamped in the forming die 218.Illustratively, only selected portions of the sheets (e.g., some or allof the portions that will be reformed) may be heated, for example bypressing the sheets between heating plates having raised portionscorresponding to the portions that will be subsequently reformed in theforming plates with the compressed fluid, as described above.

In one embodiment, liquid or aqueous reagents are injected into thereagent reservoirs 110 that are formed as described above. The aqueousreagents may be injected at the time of manufacture, or may be addedlater. Illustratively, the aqueous reagents are injected into thereagent reservoirs 104, 106, and 110 a-110 g, and the reservoirs arethen sealed to seal in the aqueous reagents. In some embodiments, one ormore of reservoirs 104, 106, and 110 a-110 g may include a dried reagent(e.g., a freeze-dried reagent pellet) that can be rehydrated at the timeof use by a liquid reagent or by liquid sample. In some embodiments,liquid reagents may be spotted onto at least one of the first and secondpolymeric sheets 200, 210 and then dried prior to laminating the firstand second polymeric sheets. Illustratively, fluids (e.g., samplepreparation fluid in the sample preparation chamber, lysis buffer, etc.)may be injected into the corresponding openings (e.g., 243 a-243 j ofFIG. 5A) that may be formed in the methods described herein.

Furthermore, a reaction card can be inserted into between the first andsecond polymeric sheets (e.g., sheets 200 and 210), such as is shown anddescribed in U.S. Pat. Pub. No. 2020/0261914, the entirety of which wasalready incorporated herein by reference. For example, a reaction card109 having a plurality of wells formed therein and spotted with one ormore dried reagents for a second stage reaction can be inserted into asecond reaction chamber 108 formed in the pouch 100 a, 100 b via anopening 244 between the first and second polymeric sheets. A firstplanar face of the reaction card may be bonded to the first polymericsheet and a second, opposite planar face of the reaction card may bebonded to the second polymeric sheet. The opening through which thereaction card was inserted can be sealed by sealing the first polymericsheet to the second polymeric sheet at the opening 244.

The present invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Whilecertain embodiments and details have been included herein and in theattached invention disclosure for purposes of illustrating theinvention, it will be apparent to those skilled in the art that variouschanges in the methods and apparatus disclosed herein may be madewithout departing from the scope of the invention, which is defined inthe appended claims. All changes which come within the meaning and rangeof equivalency of the claims are to be embraced within their scope.

What is claimed is:
 1. A method for forming a reaction container,comprising: providing a polymeric sheet that comprises an inner planarface and an outer planar face; contacting a first inner planar face ofthe polymeric sheet to a second inner planar face of the polymericsheet; pressing the polymeric sheet between a first forming plate and asecond forming plate, wherein at least one of the first or secondforming plates has one or more recesses for forming one or more featuresselected from the group consisting of a reaction chamber, a fluid flowchannel, a reagent chamber, or a sample chamber in selected portions ofthe polymeric sheet; propelling a compressed fluid between the innerplanar faces of the polymeric sheet while the polymeric sheet is pressedbetween the forming plates to reform the selected portions of thepolymeric sheet into a shape defined by the one or more recesses of theforming plates; separating the first forming plate and the secondforming plate so the polymeric sheet is no longer pressed between thefirst and second forming plates; and removing the reaction containerfrom between the first forming plate and the second forming plate,wherein the provided polymeric sheet comprises a first polymeric sheetand a second polymeric sheet, each polymeric sheet having an innerplanar face and an outer planar face, and the method further comprising:arranging the first and second polymeric sheets so that the inner planarfaces are arranged adjacent to one another, performing the pressingstep, and propelling a compressed fluid between the two polymeric sheetswhile the two polymeric sheets are pressed between the forming plates toreform the selected portions into a shape defined by the one or morerecesses of the forming plates.
 2. The method of claim 1, furthercomprising heating the polymeric sheet to a softening temperature priorto the pressing step, performing the pressing and propelling steps, andcooling the polymeric sheet after the propelling step to set the shapedefined by the one or more recesses of the forming plates.
 3. The methodof claim 1, wherein the polymeric sheet comprises a flexible polymericmaterial having a thickness in a range of about 0.02 mm to about 0.1 mm.4. The method of claim 3, wherein the flexible polymeric material isselected from the group consisting of polyester, polyethylene,polyethylene terephthalate (PET), polycarbonate, polypropylene (PP),polymethylmethacrylate, mixtures, combinations thereof.
 5. The method ofclaim 4, wherein the flexible polymeric material comprises a water vaporand/or oxygen barrier.
 6. The method of claim 5, wherein the flexiblepolymeric material with the water vapor and/or oxygen barrier has awater vapor transmission rate (WVTR) in a range of about 0.01 g/m2/24hrs to about 3 g/m²/24 hrs, preferably in a range of about 0.05 g/m2/24hrs to about 2 g/m2/24 hrs, or more preferably no more than about 1g/m2/24 hrs, and an oxygen transmission rate in a range of about 0.01cc/m2/24 hrs to about 2 cc/m2/24 hrs, preferably in a range of about0.05 cc/m2/24 hrs to about 2 cc/m2/24 hrs, or more preferably no morethan about 1 cc/m2/24 hrs.
 7. The method of claim 5, wherein theflexible polymeric material comprises two or more layers of filmmaterial bonded together and the water vapor and/or oxygen barriercomprises at least one of a metalized or ceramic-coated film layer. 8.The method of claim 1, wherein the selected portions reformed in thepropelling step comprise one or more of a sample input chamber, a samplepreparation chamber, a sample reactant recovery/wash chamber, a reactionchamber, or one or more fluid reagent reservoirs.
 9. A method forforming a reaction container, comprising: providing a first polymericsheet and a second polymeric sheet, wherein the first and secondpolymeric sheets each comprise an inner planar face and an outer planarface; contacting the inner planar face of the first polymeric sheet tothe inner planar face of the second polymeric sheet; laminating thefirst polymeric sheet to the second polymeric sheet; making one or moreseal lines joining the first and second polymeric sheets; pressing thefirst and second polymeric sheets between a first forming plate and asecond forming plate, wherein at least one of the first or secondforming plates has one or more recesses positioned for reforming thefirst and second polymeric sheets to form one or more openings in aregion defined by the one or more seal lines; expanding selected areasof the first and second polymeric sheets into a shape defined by the oneor more recesses of the forming plates by blowing a compressed gasbetween the first and second polymeric sheets while the first and secondpolymeric sheets are pressed between the forming plates; separating thefirst forming plate and the second forming plate; and removing thereaction container from between the first forming plate and the secondforming plate.
 10. The method of claim 9, wherein making the one or moreseal lines comprises defining one or more of a sample input chamber, asample preparation chamber, a sample reactant recovery/wash chamber, atleast one reaction chamber, a one or more fluid reagent reservoirs, orone or more channels fluidically connecting the sample input chamber,the sample preparation chamber, the sample reactant recovery/washchamber, the at least one reaction chamber, and the one or more reagentreservoirs.
 11. The method of claim 10, wherein when the first andsecond polymeric sheets are pressed between a first forming plate and asecond forming plate the one or more recesses of the forming platessubstantially align with the one or more areas defined by the seallines, and the selected areas expanded by blowing a compressed gasbetween the first and second polymeric sheets comprise one or more ofthe sample input chamber, the sample preparation chamber, therecovery/wash chamber, one or more reaction chambers, or one or morereagent reservoirs, and wherein one or more of the selected areasexpanded by blowing the compressed gas between the first and secondpolymeric sheets are connected by one or more sealed, openable laminatedchannels.
 12. The method of claim 11, further comprising: making the oneor more seal lines defining a second reaction chamber, expanding thesecond reaction chamber into a shape defined by a second reactionchamber recess of the forming plates, providing a reaction card having aplurality of wells formed therein and spotted with one or more driedreagents for a second stage reaction, inserting the reaction card intothe second reaction chamber via an opening between the first and secondsheets; bonding a first planar face of the reaction card to the firstsheet and a second, opposite planar face of the reaction card to thesecond sheet, and sealing the opening used to insert the reaction cardby sealing the first polymeric sheet to the second polymeric sheet atthe opening.
 13. The method of claim 11, further comprising: expanding afluid reservoir and an access channel in the first reaction chamber intoshapes defined by the recesses of the forming plates by blowing thecompressed gas between the first and second polymeric sheets while thefirst and second polymeric sheets are pressed between the formingplates, injecting an aqueous reagent into the fluid reservoir in thefirst reaction chamber via the access channel, and sealing the samplepreparation reagent in the fluid reservoir in the sample preparationchamber by sealing the first polymeric sheet to the second polymericsheet at the access channel such that the reaction container is providedwith the aqueous reagent in the first reaction chamber at the time ofmanufacture.
 14. The method of claim 1, wherein the first and secondpolymeric sheets comprise a water vapor and/or oxygen barrier providinga water vapor transmission rate (WVTR) in a range of about 0.01 g/m2/24hrs to about 3 g/m2/24 hrs, preferably in a range of about 0.05 g/m²/24hrs to about 2 g/m2/24 hrs, or more preferably no more than about 1g/m2/24 hrs, and/or an oxygen transmission rate in a range of about 0.01cc/m2/24 hrs to about 2 cc/m2/24 hrs, preferably in a range of about0.05 cc/m2/24 hrs to about 2 cc/m2/24 hrs, or more preferably no morethan about 1 cc/m2/24 hrs.
 15. The method of claim 1, furthercomprising: prior to the laminating step, dispensing droplets of one ormore liquid reagents onto the first polymeric sheet or the secondpolymeric sheet and drying the droplets of liquid reagent dispensed ontothe first polymeric sheet or the second polymeric sheet, wherein thedroplets of the one or more liquid reagents are dispensed and dried inone or more areas to be formed into the sample input chamber, the samplepreparation chamber, the sample reactant recovery/wash chamber, the atleast one reaction chamber, or the one or more channels fluidicallyconnecting the sample input chamber, the sample preparation chamber, thesample reactant recovery/wash chamber, the at least one reactionchamber, and the one or more reagent reservoirs.
 16. A method forforming a reaction container formed from a first sheet and a secondsheet and having a reaction chamber, a reagent reservoir, a channelfluidically connecting the reaction chamber and the reagent reservoir,and one or more dried reagents disposed in the reaction containerbetween the first sheet and the second sheet, the method comprising:dispensing one or more liquid reagents onto the first sheet or thesecond sheet; drying the liquid reagents dispensed onto the first sheetor the second sheet; laminating the first sheet to the second sheet,wherein the laminating includes heating the first and second sheets andcompressing them, and wherein the laminated first and second sheets arereversibly sealed to one another; forming one or more seal linessubstantially irreversibly bonding the first and second sheets togetherat the seal lines to define the reaction chamber, the reagent reservoir,and the channel; clamping the first and second sheets in a forming diehaving a first plate and a second plate, wherein the forming diecomprises a recess having a shape corresponding to the reagentreservoir; propelling a fluid between the first and second sheets whilethe first and second sheets are clamped in the forming die to reformselected areas of the first and second sheets into the shapes of therecesses; and removing the reaction container from the forming die. 17.The method of claim 16, wherein the liquid reagents are dispensed ontothe first or second sheet as droplets.
 18. The method of claim 16,wherein the liquid reagents are water-based.
 19. The method of claim 16,wherein the liquid reagents are air dried on the first or second sheetprior to the laminating.
 20. The method of claim 16, wherein the one ormore liquid reagents are dispensed onto the first or second sheet anddried in a region to be formed into the reaction chamber.
 21. The methodof claim 16, further comprising injecting an aqueous fluid reagent intothe reagent blister via an opening between the first and second sheets,sealing the fluid reagent in the reaction container by sealing theopening such that the reaction container is provided with the fluidreagent at the time of manufacture.
 22. The method of claim 21, whereinthe aqueous reagent is configured for rehydrating the one or more driedreagents disposed in the reaction container in preparation for or duringperforming an assay using the reaction container.
 23. The method ofclaim 16, wherein the first sheet to the second sheet comprise aflexible polymeric material selected from the group consisting ofpolyester, polyethylene, polyethylene terephthalate (PET),polycarbonate, polypropylene (PP), polymethylmethacrylate, mixtures,combinations thereof, and further comprising a water vapor and/or oxygenbarrier providing a water vapor transmission rate (WVTR) in a range ofabout 0.01 g/m2/24 hrs to about 3 g/m2/24 hrs, preferably in a range ofabout 0.05 g/m2/24 hrs to about 2 g/m2/24 hrs, or more preferably nomore than about 1 g/m2/24 hrs, and an oxygen transmission rate in arange of about 0.01 cc/m2/24 hrs to about 2 cc/m2/24 hrs, preferably ina range of about 0.05 cc/m2/24 hrs to about 2 cc/m2/24 hrs, or morepreferably no more than about 1 cc/m2/24 hrs.
 24. The method of claim16, wherein the liquid reagents dispensed onto the first or second sheetcomprise an enzyme selected for use in a molecular biological orimmunological assay.
 25. The method of claim 24, wherein the enzymeregains its activity following the drying, laminating, and rehydration.